CN1596304A - protein nodule - Google Patents

Abstract

Compositions comprising a knob attached to a specific location of a protein and methods of making and using the same are disclosed. These compositions include a knob, a tail, and a protein moiety. The protein moiety contains a substituted cysteine residue at the desired label. The tail is located at the end of the protein portion. The knob is attached to the end of the tail and contains a cysteine residue. The substituted cysteine residues of the protein moiety form disulfide bonds with cysteine residues on the knob, thereby labeling the protein with the knob where desired.

Description

protein nodule

Government interests

This research was supported by grant numbers NICHD HD14907 and NICHDHD38547 from the National Institutes of Health. This invention was made with United States Government support. The government may have certain rights in this invention.

field of invention

The present invention relates to the field of protein labeling.

introduction

Current methods of protein labeling for analysis of protein-protein interactions or for protein purification include fusing labels to the carboxy- or amino-terminus of proteins, or inserting residues in loops of proteins. These methods have certain limitations. For example, whether using a dye molecule or a protein, it may be more desirable to label at a specific location on the protein than simply attaching the molecule to the end of the protein. These include studies designed to probe interactions between proteins and macromolecules like hCG and its receptors. Additionally, labeling at the end of a protein may not be ideal when the end of the protein is involved in the function of that protein. Methods for inserting labels into protein loops are also limited, since labels typically must be relatively small (a few residues) if they are not inserted between protein domains. Sometimes it may be desirable to attach probes of different sizes to the protein surface. In addition, modification using cysteine residues is also very difficult because the protein may contain other cysteine residues that need to be protected or blocked cysteines, which may denature the protein when the blocking residue is removed. Therefore, the industrial application of tagged proteins produced by these methods is limited.

For example, because of the complex structure of human chorionic gonadotropin (hCG) and the similarities in its interaction with the luteinizing hormone receptor (LHR) at multiple sites, it has been difficult to identify where the hormone contacts the LHR receptor. parts. The crystal structure of human chorionic gonadotropin (hCG) shows that one strand of its beta subunit surrounds the alpha subunit like a "seat belt" (Lapthorn, A.J., Harris, D.C., Littlejohn, A., Lustbader, J.W., Canfield, R.E., Machin, K.J., Morgan, F.J. & Isaacs, N.W. (1994) Nature 369, 455-461; Wu, H., Lustbader, J.W., Liu, Y., Canfield, R.E. & Hendrickson, W.A. (1994) ) Structure 2, 545-558.). Unlike most dimeric proteins, which are completely stabilized by mutual contact between subunits, hCG seems to be mainly fixed by its safety belt; The removal of the disulfide bond of Cys26 in the basal core may disrupt hCG secretion by destabilizing the heterodimer (Suganuma, N., Matzuk, M.M. & Boime, I. (1989) J.Biol.Chem.264, 19302-19307). The evolutionary advantage of this unusual structural rearrangement remains unclear, and it may reflect a phenomenon observed when some hCG analogs bind to the FSH receptor—permitting movement of subunits in the heterodimer (Wang , Y.H., Bernard, M.P. & Moyle, W.R. (2000) Mol. Cell. Endocrinol. 170, 67-77.).

Receptors for all three classes of glycoprotein hormones, including hCG, are coupled to G proteins and have a large extracellular domain containing multiple leucine-rich repeats (Segaloff, D.L. & Ascoli, M.( 1993) Endocr. Rev. 14, 324-347.). The latter finding suggests that the extracellular domain may be partly similar to other leucine-rich repeat proteins in a horseshoe shape (Kobe, B. & Deisenhofer, J. (1993) Nature 366, 751-756). Two regions of the extracellular domain appear to be associated with the affinity and specificity of its ligand binding. The affinity of HCG for alternatively spliced and truncated LHR analogs is similar to that for the intact receptor, suggesting that the amino acid residues in the amino-terminal two-thirds of the extracellular domain of human LHR form a high-affinity binding site (Braun et al. , T., Schofield, P.R. & Sprengel, R. (1991) EMBO. J.10, 1885-1890; Thomas, D., Rozell, T.G., Liu, X. & Segaloff, D.L (1996) Mol. Endocrinol.10 , 760-768.), the finding that amino acid residues in the carboxy-terminal fifth of the extracellular domain interfere with its binding to nonhuman mammalian luteinizing hormone suggests that this portion of the hormone actually contacts the receptor The contact is mainly in space (Bernard, M.P., Myers, R.V. & Moyle, W.R. (1998) Biochem. J.335, 611-617.).

The surfaces on which hCG, hFSH, and hTSH most likely come into contact with LHR, HSHR, and THSHR remain controversial. The carboxyl terminus of the α-subunit adjacent to a partial safety zone in the heterodimer has been found (Wu, H., Lustbader, J.W., Liu, Y., Canfield, R.E. & Hendrickson, W.A. (1994) Structure 2, 545- 558.) affects the affinity of all glycoprotein hormones to their receptors (Lapthorn, A.J., Harris, D.C., Littlejohn, A., Lustbader, J.W., Canfield, R.E., Machin, K.J., Morgan, F.J. & Isaacs, N.W. (1994) Nature 369, 455-461; Bernard, M.P., Myers, R.V. & Moyle, W.R. (1998) Biochem.J.335, 611-617), and was proposed as receptor contact more than 25 years ago (Lapthorn, A.J., Harris, D.C., Littlejohn, A., Lustbader, J.W., Canfield, R.E., Machin, K.J., Morgan, F.J. & Isaacs, N.W. (1994) Nature 369, 455-461.). Taking structural and functional data for the hormone together, these findings lead to a different view of the hormone-receptor complex from first principles (Moyle, W.R., Campbell, R.K., Rao, S.N.V., Ayad, N.G., Bernard, M.P., Han , Y. & Wang, Y. (1995) J.Biol.Chem.270, 20020-20031). These views start from the fact that the hormone contacts the concave surface of the receptor extracellular domain (Jiang, X., Dreano, M., Buckler, D.R., Cheng, S., Ythier, A., Wu, H., Hendrickson, W.A., Tayar , N.E. & el Tayar, N. (1995) Structure 3, 1341-1353) to hormone-receptor edge contacts (Moyle, W.R., Campbell, R.K., Rao, S.N.V., Ayad, N.G., Bernard, M.P., Han, Y. & Wang, Y. (1995) J.Biol.Chem.270, 20020-20031) have. All views of the hCG-LHR complex suggest that a portion of the second loop of the α subunit faces the receptor, but whether this portion of the hormone is involved in receptor contact remains to be determined. It has been reported that this loop mutation will reduce the activity of hCG (Peng, K.C., Bousfield, G.R., Puett, D. & Ward, D.N. (1996) Journal of Protein Chemistry 15,547-552; Xia, H., Chen, F. & Puett, D. (1994) Endocrinol.134, 1768-1770), suggesting that it may contribute to the necessary LHR contact.

Protein-receptor interactions are key to understanding the function of cells and the regulation of their behavior. Much work, such as designing new drugs, requires an in-depth understanding of protein-receptor interactions. However, there are still many limitations in understanding protein-receptor interactions. It would be advantageous to understand the structure and function of the protein conformation, and how the structure of the protein interacts with the receptor, which is often also a protein. An aspect of a protein can sometimes be inferred through knowledge and experience if other aspects of the protein are known. Protein-receptor interactions can be simulated in silico, but the simulation is a complex task, especially since molecules are deformable and adopt a large number of energetically similar conformations. Simulation of the protein-receptor binding process is also difficult because of the need to take into account the properties of receptors, ligands, and solvents.

In addition to simulations, another approach to understanding protein-receptor interactions is to vary the protein structure and the conditions in the receptor structure and experimental setup, and measure the effect on altered binding as well as on altered or lost protein function. These experimental techniques also have limitations due to the difficulty of manipulating the protein or receptor and the limited ability to measure these changes. An invention that would improve the accuracy of experimental data by consistently inducing or inhibiting protein-receptor interactions would therefore be a major advance in the field as it would allow the determination of the resulting function or loss of function.

In principle, it should be easy to elucidate the parts of the hormone and receptor that interact with each other using site-directed mutagenesis techniques. Unfortunately, mutations in the carboxyl terminus of the α-subunit and the safety band of the β-subunit alter the position of the subunit in the heterodimer (Jiang, X., Dreano, M., Buckler, D.R., Cheng, S., Ythier, A., Wu, H., Hendrickson, W.A., Tayar, N.E. & el Tayar, N. (1995) Structure 3, 1341-1353; Pierce, J.G. & Parsons, T.F. (1981) Annu. Rev. Biochem.50 , 465-495), making it difficult to explain the influence of these key parts of the hormone on its function. Furthermore, mutations may alter critical LHR contacts, shift subunit positions, or both, resulting in altered hCG activity. Indeed, mutations at the carboxy-terminus of the α-subunit that have a major impact on hormone-receptor interactions (Pierce, J.G. & Parsons, T.F. (1981) Annu.Rev.Biochem.50, 465-495, Chen, F., Wang, Y. . & Puett, D. (1992) Mol.Endocrinol.6, 914-919) and seat belt mutation (Campobell, R.K., DeanEmig, D.M. & Moyle, W.R. (1991) Proc.Natl.Acad.Sci. (USA) 88 , 760-764; Campbell, R.K., Bergert, E.R., Wang, Y., Morris, J.C. & Moyle, W.R. (1997) Nature Biotech.15, 439-443; Grossmann, M., Szkudlinski, M.W., Wong, R. , Dial, J.A., Ji, T.H. & Weintraub, B.D. (1997) J.Biol.Chem.272, 15532-15540; Lindau-Shepard, B., Roth, K.E. & Dias, J.A. (1994) Endocrinol.135, 1235- 1240) will also change the position of the subunit in the heterodimer (Wang, Y.H., Bernard, M.P. & Moyle, W.R. (2000) Mol. Cell. Endocrinol.170, 67-77; Moyle, W.R., Campbell, R.K. , Rao, S.N.V., Ayad, N.G., Bernard, M.P., Han, Y. & Wang, Y. (1995) J. Biol. Chem. 270, 20020-20031). Another alternative approach to identifying portions of a protein that interact with a receptor involves identifying residues that do not contact the receptor. These residues can be more positively identified. However, obtaining probes to identify these residues has been somewhat difficult. Correspondingly, methods for producing probes to analyze protein interactions need to be improved.

In addition to being used to analyze protein interactions, probes or proteins labeled at specific locations on the molecule provide powerful research tools. However, as previously mentioned, the current methods of attaching or labeling probes to the termini of molecules limit their use. Other methods involve complex procedures requiring various reactive and protecting groups to generate site-directed modifications without altering other amino acids and the structure of the functional protein. Correspondingly, an improved site-specific labeling method is also required.

Protein purification is often a necessary but somewhat onerous step. A purified protein may be a necessary intermediate or end product of a scientific experiment. Protein purity is often critical to the success of trials and treatments. In some cases, injectable proteins are reviewed by the Food and Drug Administration, which must remove any contaminants or be shown to be harmless. In addition to purity, proteins must retain their biological activity.

There are many protein purification methods known in the art, although all methods have certain limitations. Size-exclusion chromatography facilitates a general separation, but is not a rigorous method and requires concentration of the sample. Gel electrophoresis can precisely separate proteins in a mixture, but is only practical for small samples. Affinity chromatography is a useful method, but often requires filtration of starting contaminants. Accordingly, an invention that would facilitate the precise separation of proteins using a small number of steps would be a significant advance in the art, especially if the invention would maintain purity while expanding purification capacity.

In addition to these uses, knobs can be used to "coat" specific surfaces of proteins. Such uses could be applied in the design of prodrugs that can be used to target tumors or other excess tissue, such as that found in the ovaries of infertile women diagnosed with polycystic ovary syndrome. In this way, linking the nodule near the active site of the toxin or toxic enzyme allows the use of the toxic enzyme in the patient. Once the toxin or enzyme reaches a certain site, the enzyme contained in this site can cleave the linker with the nodule, so that the activity of the toxin or enzyme can be restored. This strategy is expected to reduce unwanted side effects that might otherwise limit the use of the toxin or enzyme. Likewise, nodules can be used to block the activity of agents such as PTEN, which promotes apoptosis.

Summary of the invention

The present invention relates to compositions comprising proteins tagged with nodules at specific sites, their preparation and methods of use. Nodules refer to protein markers that can be tailored for specific uses.

In one aspect of the invention, proteins tagged with nodules at specific sites are disclosed. Site-specific tagged proteins include knots, tails, and protein parts. Nodules contain marker or probe aspects and have cysteine residues. The tail is located between the knot part and the protein part. The protein part has a cysteine residue in place of the original amino acid where labeling is desired. The cysteine residues of the nodule form disulfide bonds with the cysteine residues of the protein moiety. In another aspect of the invention, the tail may comprise a protease or other cleavage site.

In another aspect of the invention, methods for the production of proteins site-specifically labeled with nodules are disclosed. These methods include selecting the protein to be labeled, selecting the labeling site on the protein to be labeled, and generating the desired protein; wherein the site to be labeled in the desired protein is replaced with a cysteine residue, and the desired protein A tail is also included at one end and contains a knot at the end of the tail, wherein the knot contains a cysteine residue that forms a dichotomy with a cysteine residue in the protein portion. sulfur bond. In a related aspect of the invention, methods of linking nodules to specific sites on hCG are disclosed. These methods include co-expression of constructs expressing native hCGβ or hCGβ-S138C, native hCGα, or hCGα-cysteine surrogate analogs into cells, and fusion of the nodule to residues 140 or 145 of hCGβ .

In another aspect of the invention, methods for purifying proteins using site-specifically tagged proteins of the invention are disclosed. These methods involve introducing into cells a construct encoding a protein that includes a cysteine residue substituted at the site that needs to be labeled, a cysteine residue at one end of the protein, and a cleavage The tail of the site, and the nodule portion at the end of the tail; conditions are provided to allow expression of the construct; cells are lysed and the protein is purified based on the characteristics of the nodule on the protein.

In another aspect of the invention, methods of using protein nodules for site-directed modification are disclosed. For example, protein nodules can be used to map distances between proteins, probe the surface of protein-protein interfaces, form complexes between two unrelated proteins, probe protein structure and function of protein nodules, combine proteins Immobilized on the surface, the protein is delivered to the cell as the target protein, and it can also be used for protein purification. Those skilled in the art will recognize that proteins tagged at specific sites with custom markers are powerful research tools with broad utility.

Brief description of the drawings

Figure 1. Three-dimensional view of hCG. The hCG alpha subunit residues that can be scanned by the carboxy terminus of the hCG beta subunit are shown. The backbones of the α and β subunits are represented by black and light gray bands, respectively. The β tail is indicated by a black band. The position of the Cα carbon atom in the alternative cysteine residue that allows efficient crosslinking between the α subunit residue and the probe cysteine is indicated by a black ball. Lighter gray balls refer to residues that produced less cross-linking. Gray globules indicate cysteine substitutions with negligible amounts of cross-linking. NOTE: The motility of residues 90, 91 and 92 of the α subunit appears to be too strong to find them in the crystal structure of hCG, the approximate positions of these residues are shown here only to emphasize their apparent The ability to lock the seat belt.

Figure 2A. Binding of hCG or hCG analogues wherein a native residue in the second loop of the alpha subunit of the analogue is replaced by cysteine.

Figure 2B. cAMP accumulation of hCG or hCG analogues in which a native residue in the second loop of the alpha subunit of the analogue is replaced by cysteine.

Figure 3A. Effect of substitution of alpha subunit carboxy-terminal cysteine. Conjugation of hCG or an hCG analog in which one residue at the carboxy-terminal end of the alpha subunit of the analog is replaced by cysteine.

Figure 3B. Effect of substitution of alpha subunit carboxy-terminal cysteine. Accumulation of cAMP by hCG or hCG analogues in which one residue at the carboxy-terminal end of the α-subunit of the analogue is replaced by cysteine.

Figure 4A. Binding of hCG or hCG analogues containing a beta tail attached to the second loop of the alpha subunit.

Figure 4B. cAMP accumulation of hCG or hCG analogs containing an alpha tail attached to the second loop of the alpha subunit.

Figure 5A. Binding of hCG or hCG analogs containing a beta tail attached to the carboxy-terminal residue of the alpha subunit.

Figure 5B. cAMP accumulation of hCG or hCG analogs containing a beta tail attached to the carboxy-terminal residue of the alpha subunit.

Fig. 6. Binding and signaling activity of analogs, wherein the α-subunit residues of the analogs are linked with BLA.

Figure 7. Amino acid sequences of the alpha subunit and mutants containing substituted cysteines. (Note: The mutations are shown in capital letters and highlighted. These mutants were prepared by conventional cassette mutagenesis and PCR mutagenesis methods in the art.) (SEQ ID NO: 1～SEQ ID NO: 35).

Figure 8. Amino acid sequences of β subunit analogs. (Note: replace cysteine with capital letters and highlight.) (SEQ ID NO: 36～SEQ ID NO: 42).

Figure 9A. Protein Knob, where the Knob is attached to the end of the carboxy-terminus of the protein.

Figure 9B. Protein nodules where the nodules are attached to the end of the amino terminus of the protein.

Figure 10A. Protein nodules, wherein the nodules comprise cysteine residues.

FIG. 10B . Protein nodules, wherein the nodules include amino acid sequences fused to proteins containing cysteine residues.

FIG. 10C . Protein nodule, wherein the cysteine residue of the nodule is located on the surface of the nodule protein.

Figure 11. Effect of nodules on FSH activity.

Figure 12. Summary of activity of cross-linked chimeric analogues in LHR and FSHR assays relative to CF101-109, a bifunctional chimera that does not contain the carboxyl terminus of the hCGβ subunit.

Figure 13. Effect of nodules on FSH receptor signaling.

Figure 14A. Effect of nodules on LH receptor signaling.

Figure 14B. Effect of nodules on LH receptor signaling.

Figure 15. Amino acid sequences of other analogs (SEQ ID NO: 43-SEQ ID NO: 53).

Figure 16. Signaling and binding activity of long and short heterodimers.

Figure 17. Luteinizing hormone activity of hCG analogues containing β-lactamase nodules.

Figure 18. cAMP accumulation of hCG and analogs lacking the αAsn52 oligosaccharide.

Figure 19. Binding of αK44A+hCGβ to LHR.

Figure 20A. Binding of hCG and hCG analogs αK44E, K45Q+hCGβ to LHR.

Figure 20B. LHR cAMP in response to hCG and αK44E, K45Q+hCGβ.

Figure 21A. Binding of hCG and hCG analogs αK91E+hCGβ to LHR.

Figure 21B. Relative activity of hCG and αK91E+hCGβ in LHR cAMP accumulation assay.

Figure 22A. LHR cAMP in response to hCG and αK91M+hCGβ.

Figure 22B. Binding of hCG and αK91M+hCGβ to LHR.

Figure 23. Binding of hCG and analogs containing truncated linkers to LHR.

Figure 24. Binding of hCG and analogs containing truncated linkers to LHR.

Fig. 25. Stimulatory effect of hCG and αN52C+hCGβ, S138C on LHR cAMP.

Figure 26. Binding of analogues to LHR, the carboxy-terminus of the α-subunit of the analogue adds a knot to residue 96, 97 or 99 of the hCGβ-subunit with a truncated hCGβ-subunit carboxyl tail.

Figure 27. LHR signal transduction of analogues, the carboxyl terminus of the α subunit of the analogue is truncated to the 98th or 99th residue of the hCGβ subunit with a truncated hCGβ subunit carboxy terminus.

Figure 28. Binding of analogs to LHR in which a truncated hCG beta subunit carboxyl tail is used to add a nodule to residue 95 or 96 of the beta subunit.

Figure 29. LHR signaling of analogs in which a truncated hCGβ subunit carboxyl tail is used to add a nodule to residue 95 or 96 of the hCGβ subunit.

Figure 30. Compared with analogs with missing tails and analogs with a truncated hCGβ subunit carboxyl tail to add a nodule to residue 96 of the hCG/hFSH chimera β subunit, using the GGC tail to LHR signaling by analogues with a knob added to residue 96 of the β subunit.

Figure 31. LHR signaling of analogs in which a truncated hCG beta subunit carboxyl tail is added to residues 98 or 99 of the beta subunit with a nodule.

Figure 31. LHR signaling of analogs showing the effect of the tail on the addition of a nodule to residue 95 of the hCG beta subunit, and for an analog that does not have the ability to glycosylate residue 52 of the alpha subunit Impact.

Figure 32. Cyclic AMP in response to cross-linked analogs.

Detailed description of the invention

At present, most of the labeled proteins are prepared in the form of fusion proteins by linking labels to the amino-terminal or carboxy-terminal ends of proteins, or inserting a few residues into protein loops. Although end tags can be of any size, tags inserted into protein loops are generally limited to a relatively small number of amino acid residues, except where tags are inserted between protein domains. Proteins can be labeled at different positions by introducing cysteine at the position to be labeled, and then reacting the sulfhydryl group of cysteine with a sulfhydryl-specific reagent. However, this is very difficult to accomplish when the protein contains other cysteines or is produced under conditions that result in the cysteines being blocked, which is often the case when proteins are expressed in eukaryotic cells.

The present invention provides improved methods of labeling proteins. The method of the present invention avoids the complications associated with the introduction of cysteines for labeling as described above, allowing probes or nodules of different sizes to be attached to the surface of the protein in addition to the ends of the protein. The present invention provides methods for attaching "nodules" of different sizes to specific locations on the surface of hCG. Nodules can be as small as a single cysteine residue. The nodules may be short peptides, such as the residues surrounding residue 138 of the hCGβ subunit. Nodules can also be as large as whole proteins, eg β-lactamases can be used as nodules. The nodules are added during protein synthesis so that it is not necessary to remove any blocking residues or add any protecting groups that would result in protein denaturation.

The flexible β-subunit tail can be cross-linked to one of several α-subunit residues, one of these residues being replaced by a cysteine; based on this discovery the present invention employs a new strategy to attach a broad range of probes or labels to specific locations on protein surfaces. Nodules can be fluorescent proteins such as GFP or other related molecules. They have the characteristics of ligands or receptors and can bind to other molecules. They may be proteases, toxins, antibodies or antibody fragments, sequences enabling transmembrane translation of proteins as found in the human immunodeficiency virus TAT protein, nucleic acids or oligosaccharides.

In one embodiment, the composition comprises a protein moiety, a tail at the end of the protein moiety, and a knob moiety; wherein the protein moiety contains a substituted cysteine at the location to be labeled, and the knob is located at the free end of the tail and contains a cysteine amino acid residues, the cysteine residues of the nodule form disulfide bonds with the alternative cysteine residues of the protein moiety. The term "protein moiety" refers to any protein or polypeptide. The term "tail" refers to a sequence of amino acids long enough to allow a cysteine in the knot to form a disulfide bond with an alternate cysteine in the protein portion. The tail may include part of the native polypeptide of the protein in the protein part, as in the carboxyl terminus of the beta subunit in hCG (see Figure 1), and may also include a non-native polypeptide linked to the end of the protein part. The tail should also be free of residues that would prevent the nodule from being attached to an alternative cysteine, such as a transmembrane domain or create a site that can be bound by other proteins. The term "knot" refers to a cysteine and the residues on either side of the cysteine, where the cysteine residue is adjacent to the free end of the tail. Nodules may consist of a single cysteine residue, a linear amino acid sequence containing one cysteine, a linear amino acid sequence fused to a protein with the cysteine located within the sequence, or cysteine residues on the surface base protein. Nodules can be engineered for specific purposes or uses, resulting in custom markers or probes. For example, a nodule can be a phenotypic marker, a signal sequence, a sequence with high specificity for magnetic beads embedded in a purification column, an enzyme, or a target protein.

In another embodiment of the invention, a method of labeling a protein at a specific site is disclosed. The method includes selecting the protein to be labeled, selecting the labeling site on the desired protein, and selecting the desired node. Desired nodules must contain a cysteine residue. The method also includes making constructs encoding the desired protein, the tail, and the desired knot. The desired protein encoded by this construct includes a substituted cysteine residue at the desired label. This construct is then introduced into cells, allowing expression of the tagged protein in which the cysteine in the nodule and the cysteine of the desired protein form a disulfide bond.

The term "construct" refers to a nucleic acid vector comprising a promoter linked to an expression cassette encoding a specific protein product. The construct also includes all necessary sequences to enable expression of the encoded protein, as well as any sequences that can regulate the expression of the expression cassette. These sequences include, but are not limited to, promoter or initiation sequences, enhancer sequences, termination sequences, RNA processing signals, and/or polyadenylation signal sequences. The term "sequence required for the expression of the expression cassette" refers to the sequence required to ensure that the expression cassette produces a protein product during RNA transcription and subsequent translation. The term "promoter" refers to a DNA sequence that RNA polymerase can bind to and initiate RNA transcription of a gene. There are many promoters well known in the art, including those that enhance or control the expression of a gene or expression cassette. The constructs of the invention can be modified using PCR and cassette mutagenesis techniques to generate constructs encoding desired protein nodules.

Knobs located at specific positions in a protein can be used to probe the distance of a test residue from a receptor or other protein interface. For example, nodules can be used to determine the proximity of the alpha subunit of hCG to the receptor binding site. Attachment of the probe to residues located in the binding pocket will disrupt binding activity, suggesting that the label is located within the binding pocket. Another advantage of using site-directed nodes is that larger probes can be used to identify residues near the protein-receptor interface. This probing strategy can also be used to attach phenotypic markers or signal sequences to any desired site on the protein surface. In addition, protein probes can also be widely used in protein immobilization and protein targeting. If the protease recognition site is designed on a deformable tail, the tail can be cleaved off after cross-linking, allowing the probe to be tethered by disulfide bonds on the protein surface rather than by the tail.

The use of protein probes generated by the methods of the present invention is not limited to inferring distances between protein sites. If you need to target protease, you only need to slightly modify the existing fusion protein preparation method (Sledziewski et al., US Patent No. 6,300,099), and simply fuse the coding sequence of protease to the 5' or 3' end of the protein coding sequence, A number of different proteases can then be attached to the amino or carboxyl terminus of the protein. Unfortunately, the proximity of proteases to a protein destroys that protein.

Using a protein nodule solves this problem because using the strategy described here for attaching a nodule to a protein, the protease can be fixed in a position where it cannot attack the attached molecule. Furthermore, protease orientation can catalyze the cleavage of desired substrates, such as receptors. A protease can be engineered onto hCG using the methods described herein so that it cleaves the LH receptor in preference to any other protein, rendering it incapable of luteinizing hormone activity. This protease protein nodule could serve as an ideal therapy in the treatment of polycystic ovary syndrome, which is responsible for nearly one-third of all human infertility.

In another embodiment of the invention, the compositions and methods of the invention can be used to promote the stable association of two proteins. The data presented here show that cross-linked hCG protein analogs are much more stable than native hCG at low pH. The introduction of disulfide bonds between hCG subunits increases the stability of heterodimers [Matzuk, M.M. & Boime, I. (1988) J. Cell Biol.106, 1049-1059; Heikoop, J.C.; van, den boogaart Mulders, J.W.; Grootenhuis, P.D., (1997), Structure-based design and protein engineering of intersubunit disulfide bonds in luteinizing hormone-releasing hormone, Nature Biotechnology 15:658-662]. Previously, intersubunit disulfide bonds have been introduced into proteins based on crystal structure. The present invention discloses methods for introducing intersubunit disulfide bonds between two proteins during protein synthesis when high resolution structures are not available.

In another embodiment of the invention, the compositions and methods are useful for promoting the stable association of DNA polymerases with DNA. It is expected that the introduction of a linker that wraps around the DNA and immobilizes the polymerase with a disulfide bond stabilizes the polymerase to the DNA, thereby increasing the length of the final transcript.

In another embodiment of the invention, the complexes and methods of the invention can be used to generate protein heterodimers composed of glycoprotein hormones lacking one or more oligosaccharides. It was hoped that the loss of the glycosylation signal in the second loop of the hCG α subunit would reduce the ability of mammals to secrete heterodimers, stimulating biological responses (Einstein, M., Lin, W., Macdonald, G.J. & Moyle, W.R. (2001) Exp.Biol.Med.226, 581-590; Slaughter, S., Wang, Y.H., Myers, R.V. & Moyle, W.R. (1995) Mol. Cell. Endocrinol.112, 21-25; Yen, S.S.C. Matzuk, M.M.; Boime, I., (1989), Mutagenesis and Gene Transfer Role of specific sites in defining luteinizing hormone releasing hormone oligosaccharides, Biol. Reprod. 40:48-53). As shown here, co-expression of hCG βS138C and αN52C, an α-subunit analog in which the Asn52 residue is replaced by cysteine, resulted in yields of heterodimers comparable to hCG. This alpha subunit mutation eliminates the glycosylation signal, resulting in a much more efficient hCG analog than hCG analog in which the oligosaccharide in the second loop of the alpha subunit is digested by glycanase.

In another embodiment, methods of utilizing the compositions and methods of the invention to facilitate the formation of protein multimers in which subunits have little or no affinity for each other are disclosed. For example, the data presented in Figure 6 demonstrate that it is possible to attach a beta-lactamase to one of several different sites in hCG, whereas beta-lactamase is not known to be associated with hCG. By introducing a protease cleavage site (cutting the carboxyl terminal of the β subunit) between the β subunit of hCG and the cysteine on the safety belt, the preparation of β-lactamase or other proteins can be stably linked to almost any position of hCG heterodimers are possible.

In another embodiment, the cysteines on the nodule can be moved to a surface location on the nodule portion. This allows the nodule to attach directly to the protein in the desired orientation and location.

In another embodiment, proteins can be tagged with phenotypic markers using the methods of the invention. Phenotypic markers are often attached to proteins to facilitate detection of protein-protein or protein-macromolecule interactions. In the past, phenotypic markers were attached to the amino or carboxyl termini of proteins. However, many phenotypic markers are only effective on one end of the protein. Also, the utility of phenotypic markers added to protein ends is greatly reduced when the protein ends are involved in the function of the protein. The method of the present invention allows phenotypic markers to be located at sites other than protein termini, making the phenotypic markers more useful. The introduction of a cleavage site at the tail allows the ends of the protein to be freed without destroying the phenotypic marker.

In another embodiment of the present invention, aldehyde groups can be introduced at specific sites in the protein. Aldehyde groups are a highly desirable reactive group not normally found in proteins and can be used to attach several different reactants, such as fluorophores, to the surface of proteins. This method takes advantage of the well-known reactivity of amino-terminal serine or threonine residues to mild periodate oxidation (Yoo, J., Ji, I. & Ji, TH (1991), J. Biol .Chem.266, 17741-17743; Geoghegan, KF, Stroh, JG, (1992), Non-peptide groups undergo site-specific binding to peptides and proteins by periodate oxidation of 2-aminoethanol, serine N-terminus Applications of Modifications, Bioconjug. Chem. 3: 138-146). In this way, a serine residue can be introduced into the cleavage site of the protein immediately following enzymatic cleavage. For example, an enterokinase-recognizable site can be immediately introduced into the amino-terminal serine, allowing the amino-terminal cysteine to crosslink the nodule to the target cysteine at the site to be labeled. This would generate the sequence _X1 -Asp-Asp-Asp-Asp-Lys-Ser- _Ym -Cys- _Zn (SEQ ID NO: 56), where X, Y and Z refer to any amino acid in the tail, l, m and n Refers to the length of the tail amino acid. Production of the protein results in cross-linking of the cysteine in the tail to the desired site on the protein, where the site has been replaced with a cysteine. Cleavage by enterokinase results in a terminal serine that is susceptible to oxidation by mild periodate treatment. The resulting aldehyde group is readily reactive with a variety of hydrazide-derived compounds, including various fluorophores and biotin. This method is especially useful when the protein does not contain unincorporated cysteines.

In another embodiment, methods of using the compositions of the invention to block specific sites on proteins are disclosed. For example, it is ideal to block active sites of toxins or enzymes that could kill cells until their activity is needed. Cancer treatment may be a very good field of use of the compositions and methods of the present invention. Thus, a cysteine can be introduced at a position near the active site of the enzyme or toxin. Attaching a cysteine-containing nodule to the amino or carboxyl terminus of a protein that can form disulfide bonds with cysteines near the active site prevents the active site from interacting with its target. A nodule may include a target protein fused to a tail that anchors the enzyme/toxin-target protein complex with a specific target to the cell surface. Treatment of the complex with a protease that cleaves the tail will expose the active site of the enzyme or toxin. This strategy can be used to mask the activity of an enzyme or toxin until it reaches a site containing an enzyme that cleaves the tail, thereby exposing the toxin. This can occur, for example, following internalization of the complex into the cell.

Another purpose of the nodules is to prevent this unwanted association between proteins, which normally complex with each other. In this way, disulfide bonds can be designed to hold the nodule in place between two protein interface surfaces.

In another embodiment, the present invention provides a method for the precise isolation of a target protein using a small number of steps. Expression constructs encoding the protein of interest, tails and nodules were constructed using the methods described here. The location of the target cysteine on the protein of interest can be at the carboxy-terminus, amino-terminus, or any desired site on the surface of the protein of interest. After expression of the construct, once the conjugated cysteine on the nodule binds to the cysteine on the desired protein, the resulting protein-nodule complex is rapidly passed through the column. By selecting suitable tight-binding nodules, the protein-nodules bind to the affinity resin and unbound proteins and cellular components are eluted. Then, the complex is eluted and the nodules are excised, leaving only the purified protein. For example, (Strategene's Affinity ^™ ) pCAL vector can be used, with calmodulin binding peptide (CBP) selected as the node. The CBP-nodule is bound to an affinity resin and can be eluted with 2mM EDTA at neutral pH, avoiding harsh elution conditions that may denature the protein. However, the present invention can utilize many possible vector and nodule combinations.

In another aspect of the invention relating to protein purification, the protein of interest can be constructed so that the protein knot has a short tail containing a cleavage site. The nodule tail must be short enough that the nodule cannot form a disulfide bond with the protein itself. Instead, the structure of the protein and tail should allow the knot to form a disulfide bond with another protein. In solution, the tailed proteins will appear in a linear arrangement like a string of beads formed by the cysteines on the nodules of the first protein and the cysteines on the protein moiety of the second protein. linked by disulfide bonds. A cysteine on the nodule of the second protein forms a disulfide bond with a cysteine on the protein portion of the third protein, and so on. The resulting terminal protein chains can be subjected to sucrose gradient centrifugation and the protein chains will settle to the bottom of the gradient due to their weight. Finally, to separate the protein chains from other sedimented material, such as lysed cell membranes, the underlying layer on which the protein chains reside is treated with an enzyme that specifically cleaves short tails, yielding individual proteins. The mixture was centrifuged again. Individual proteins will park near the top of the gradient for easy collection and purification. Protein chains can also be purified by other methods described above or by protein purification methods well known in the art.

Another aspect of the invention is to construct the protein of interest so that each protein nodule forms a lattice-like structure with another protein nodule. Similar to the tails in the string of beads approach, the tails should be constructed such that the nodules cannot react with cysteines on the protein moiety. Proteins can also include tails and knots at both ends of the protein. Protein moieties should include multiple substituted cysteine residues so that multiple nodules from a single protein can form disulfide bonds with the protein. The number and location of substituted cysteines on protein nodules should be determined in advance using computer modeling programs or other methods. In solution, a single protein can form disulfide bonds with multiple proteins, resulting in a lattice-like structure. Protein nodule matrices can be purified by any of the methods described above, keeping in mind that the larger the molecular weight of the structure, the more likely it is to be a good candidate for centrifugation techniques.

In another embodiment, the present invention can be used to add cysteine to proteins. Incorporation of cysteines into proteins typically takes advantage of the unique reactivity of cysteines to attach to the surface of proteins and attach other molecules, such as fluorophores, to proteins containing another cysteine. Many proteins contain cysteines or disulfide bonds, making it difficult to utilize the cysteines introduced into the molecule. This difficulty is circumvented by introducing tryptophan into the tail immediately adjacent to the cysteine used to crosslink the nodule to the protein to be modified where the cysteine was introduced. Due to the different absorption spectra of tryptophan, tyrosine, and phenylalanine, light irradiation with a wavelength of 295 nm will selectively target tryptophan residues in proteins. This will result in the destruction of adjacent disulfides, rendering the sulfhydryl group on the cysteine reactive. Irradiating the protein in the presence of the groups to be added to the protein will label the protein where desired, even if the protein contains several other disulfides. Although this favors other disulfides near the tryptophan residue also becoming reactive, the fact that most proteins actually contain very little tryptophan means this is usually not a problem.

In another embodiment, the present invention can be used to generate and obtain high yields of heterodimeric proteins. When constructing expression constructs for protein nodules, one of the heterodimers may include the protein moiety. If the protein portion of the dimer does not have a naturally occurring tail, the tail can be fused to the protein portion of the dimer. Nodules may include other dimers of heterodimers. When the construct is expressed, there is a greater likelihood of heterodimer formation due to the presence of the linking dimer tail and the ability to form disulfide bonds. This approach avoids the problem of homodimer formation when dimers of heterodimers are co-expressed in the same cell.

Example 1 - Protein nodules to study hCG-LHR interaction

The sources of hCG and antibodies used in these studies have been described (Bemard, MP, Myers, RV & Moyle, WR (1998) Biochem. J. 335, 611-617; Moyle, WR, Campbell, RK, Rao, SNV, Ayad, NG, Bernard, MP, Han, Y. & Wang, Y. (1995) J. Biol. Chem. 270, 20020-20031; Moyle, WR, Matzuk, MM, Campbell, RK, Cogliani, E., Dean Emig, DM, Krichevsky, A., Barnett, RW & Boime, I. (1990) J. Biol. Chem. 265, 8511-8518.). A construct capable of expressing hCGβ-S138C was prepared by cassette mutagenesis between the native ApaI site and the BamHI site of hCG cDNA, wherein the BamHI site was designed downstream of the terminator as described in the literature (Campbell, RK, Dean Emig, DM & Moyle, WR (1991) Proc. Natt. Acad. Sci. (USA) 88, 760-764; Campbell, RK, Dean Emig, DM & Moyle, WR (1991) Proc. Natt. Acad. Sci. (USA) 88, 760-764.). Simultaneously, prepare the vector (Xing, Y., Lin, W., Jiang, M., Myers, RV, Cao, D., Bernard, MP & Moyle, WR, Other-folded chorionic gonadotropin analogs: hints for hormone folding and biological activity. Journal of Biological Chemistry.2001.). The construct encoding human α subunit or cysteine replacement analog was co-expressed with hCG β subunit or hCG β-S138C in COS-7 cells according to the method described in the literature (Campbell, RK, Dean Emig, DM & Moyle, WR (1991 ) Proc. Natt. Acad. Sci. (USA) 88, 760-764.). Secretion into the culture medium was determined using a sandwich immunoassay using α-subunit antibody A113 for capture and radioiodinated β-subunit antibody B110 for detection (Moyle, WR, Ehrlich, PH & Canfield, RE (1982 ) Proc. Natt. Acad. Sci. (USA) 79, 2245-2249.). It was also treated with acidic pH as described in the literature to promote the dissociation of heterodimers lacking disulfide crosslinks (Xing, Y., Lin, W., Jiang, M., Myers, RV, Cao, D., Bernard, MP & Moyle, WR Other folded analogs of chorionic gonadotropin: hints for hormone folding and biological activity. Journal of Biological Chemistry.2001.). As previously reported, Chinese hamster cells (CHO) overexpressing rat LHR were used to monitor the effect of analogues on the ability of ¹²⁵ I-hCG to bind to LHR and stimulate the accumulation of cyclized AMP (Bernard, MP, Myers, RV & Moyle, WR (1998) Biochem. J. 335, 611-617; Moyle, WR, Matzuk, MM, Campbell, RK, Cogliani, E., Dean Emig, DM, Krichevsky, A., Barnett, RW & Boime, I. (1990) J. Biol. Chem. 265, 8511-8518; Moyle, WR, Campbell, R. K., Myers, RV, Bernard, MP, Han, Y. & Wang, X. (1994) Nature 368, 251-255.).

An alternative approach to explain the hCG-LHR interaction involves identifying the exposed surfaces of this hormone in the hormone-receptor complex (Moyle, W.R., Matzuk, M.M., Campbell, R.K., Cogliani, E., Dean Emig, D.M., Krichevsky , A., Barnett, R.W. & Boime, I. (1990) J. Biol. Chem. 265, 8511-8518.). By exclusion, exposed regions of the hormone-receptor complex were mapped to the surface of the hormone to reveal sites of accessible receptor contact. Since these data were collected in studies in which the hormone retained its receptor-interacting activity, it is easier to interpret than data that depend on altered hormone-receptor interactions. Most methods for detection of noncontact residues rely on the use of monoclonal antibody probes, which are severely limited by resolution. To circumvent this limitation, the activity of analogues locked by seat belts can be determined (Xing, Y., Lin, W., Jiang, M., Myers, R.V., Cao, D., Bernard, M.P. & Moyle, W.R. Other-folded chorionic gonadotropin analogs: Implications for hormone folding and biological activity. Journal of Biologicalchemistry.2001.). Several of these analogs have essentially the same activity as hCG, although containing part of the safety belt and the carboxy-terminal linkage region may block hormone receptor interactions. Linking the safety belt to the α subunit may change the conformation of the dimer, which may explain the low activity of some analogs. Recently, it was found that the scrambled long carboxyl terminus in the β subunit is sufficiently mobile to scan most of the surface of the heterodimer until the introduction of a cysteine at residue 138 in conjunction with the introduction of a cysteine in the α subunit. Cysteine forms a disulfide bond. The safety belt in these analogs is tethered like the safety belt in hCG, making it difficult for the mutation to affect the conformation of the heterodimer. As described here, many of these analogs are more active than those in which the harness has been linked to the beta subunit. The results of the studies performed also showed that most of the second loop of the hCG α subunit does not contact the LHR, and that the second loop may be close to the receptor interface. We compared the activity of hCG analogues before and after the carboxyl terminus of the jumbled beta subunit was tethered by the cysteine introduced by the second loop and the carboxyl terminus of the alpha subunit (see Figures 2 and 3). Heterodimers lacking crosslinking were at least 25% as active as hCG in LHR binding and signaling assays, except for analogs with cysteine at the tip of the second loop. This result suggests that very few residues in either region contribute a fraction of the total energy of hCG-LHR binding, which is in conflict with reports that the carboxy terminus is essential for potency. Tethering the β-subunit carboxy-terminal probe to the second loop of the α-subunit facing the first and third loops of the β-subunit had relatively little effect on binding and signaling, suggesting that this part of the hormone Contact with receptors is unlikely. Linkage of the carboxyl terminus of the β subunit to other residues results in loss of activity, suggesting that these portions of the α subunit may be in proximity to the receptor. Using this method to study the interaction of hFSH with the FSH receptor revealed that hFSH binds to the FSH receptor in an overall similar manner, but that the portion of hFSH that contributes to key receptor contacts differs. These observations were also confirmed by using this method to study chimeric hCG-hFSH ligands that bind both LH and FSH receptors. Thus, different parts of the chimeras are closer to the LHR than to the FSHR. A similar mutagenesis strategy should be useful in identifying parts of other proteins that do not participate in protein-protein contacts.

The studies described here also demonstrate the use of this approach to identify the binding site of hFSH binding to the FSH receptor and compare the interaction of bifunctional hCG/hFSH analogs with the LH receptor and the FSH receptor. hFSH has no "safety belt" (tail) (Fig. 8, SEQ ID NO: 41), so a hCG β subunit carboxyl terminal (fqdsssskapppslpspsrlpgpstdpilpg, SEQ ID NO: 55) partial residue encoding at the carboxy terminus of the β subunit was prepared hFSH subunit analogs. Similar to the substitution of cysteine for serine residue 138 in the studies performed with hCG, the substitution of cysteine for serine residue 132 of the hFSH beta subunit analog (Figure 8, SEQ ID NO: 42). The hFSH beta subunit S132C was then expressed together with several alpha subunit analogs containing one substituted cysteine. As expected from experience with hCG analogs, this heterodimer is much more stable at low pH than native hFSH, demonstrating that the β and α subunits of the hFSH analogs are cross-linked via disulfide bonds. Many of these analogs were highly active in the hFSH receptor assay (Figure 13). The hFSH-derived analogs showed some differences in activity assays compared to the hCG-derived analogs. These differences suggest that HSH interacts differently with the FSH receptor and hCG interacts with the LH receptor, and thus the conformational model of each hormone-receptor is different. The results also suggest that the hCG beta subunit carboxy-terminal sequence may act as a "tail", adding a nodule to the surface of a protein that lacks a suitable site or "tail". The "tail" sequence needs to be long enough so that the cysteines in the tail can reach the cysteines that are attached to the surface of the protein, and the sequence must not contain residues that prevent the sequence from accessing the cysteines on the surface of the protein. Residues that prevent the sequence from accessing the cysteine on the surface of the protein include those that cause the sequence to fold into an insulating domain that shields the cysteine, such as residues that allow the protein to become attached to the cell membrane so that the cysteine Acid-shielded signal residues, residues containing a signal that binds a protein to another protein thereby shielding the cysteine, or highly charged residues that block interactions between protein surfaces.

Bifunctional analogs of glycoprotein hormones can be prepared by interchanging portions of the harness of glycoprotein hormones (Moyle et al., US Patent No. 5,508,261). In order to further distinguish the interaction between luteinizing hormone, such as the difference between hCG and LH receptors; and the difference between follicle-stimulating hormone interactions, such as the difference between hFSH and FSH receptors, we prepared a protein that is known to interact with both LH and FSH receptors. An hCG analog that binds to the body. Residues 101-114 of hCG were replaced with their hFSH counterparts, residues 95-108 of hFSH (FIG. 8, SEQ ID: 38). As in the previous hCG and hFSH analogues, the 38th serine residue at the carboxyl terminus of this analogue was replaced with cysteine (FIG. 8, SEQ ID: 39). The constructs were expressed in COS-7 cells together with several analogs containing substituted cysteines. The resulting heterodimer was quantified using a sandwich immunoassay using antibodies to the alpha and beta subunits of hCG. Based on the increased stability at low pH, many heterodimers were found to be cross-linked via disulfide bonds between the α and β subunits. Some of these analogs interact significantly differently than the interaction between LH and FSH receptors. For example, an analogue cross-linked at the carboxyl terminus of the β subunit to cysteine residue 37 of the α subunit interacts well with the LH receptor but not adequately with the FSH receptor (Fig. 12-14). This confirms that the interaction of hCG with the LH receptor is distinct from the interaction of hFSH with the FSH receptor and provides important further support for models of the interaction of individual hormones with their receptors.

result

hCGα subunit analogs vs native hCGβ subunit or hCGβ-S138C

COS-7 cells co-transfected with vectors encoding most of the α subunit analogs and vectors encoding the native β subunit (Table 1) or hCGβ-S138C (Table 2) can assemble heteromers and secrete them into culture Base. Heterodimers secreted with little or no secretion included those analogs in which cysteine was substituted for Tyr37, Pro40, Asn52, and Y89C residues in the beta and alpha subunits (Table 1). The minimal secretion of αN52C/β likely reflects its lack of an N-linked glycosylation signal normally required for efficient secretion of the heterodimer (Matzuk, M.M. & B, I. (1988), J. Cell Biol. 106, 1049-1059).

Table 1. Heterodimer production in COS-7 cells transfected with the indicated subunit constructs transfection Dimer content in medium (ng/50μl) mean±SEM αG22C+β 9.034 ± 0.414 αY37C+β 0.464 ± 0.052 αP38C+β 1.710 ± 0.026 αP40C+β 0.588 ± 0.093 αL41C+β 7.617 ± 0.438 αR42C+β 0.962 ± 0.135 αS43C+β 3.571 ± 0.156 αK44C+β 2.788 ± 0.358 αK45C+β 1.090 ± 0.093 αT46C+β 7.044 ± 0.229 αM47C+β 9.491 ± 0.524 αL48C+β 1.497 ± 0.170 αV49C+β 2.579 ± 0.297 αQ50C+β 1.985 ± 0.153 αN52C+β 0.615 ± 0.081 αV53C+β 6.108 ± 0.356 αM71C+β 6.153 ± 0.332 αG73C+β 2.849 ± 0.194 αT86C+β 4.973 ± 0.027 αY88C+β 7.930 ± 0.290 αY89C+β not detected αH90C+β 0.971 ± 0.170 αK91C+β 1.049 ± 0.055 αS92C+β 2.595 ± 0.106

Most of the α-subunit analogues were detected in heterodimers containing hCGβ-S138C (Table 2). hCGβ-S138C promotes the formation of several analogs that are minimally secreted when co-expressed with the native β subunit, including Tyr37, Pro40 and Asn52, but not Tyr89 (Table 2, Figure 1). Many of the analogs in Table 2 exhibit intersubunit cross-linking, which is easily detected by simple treatment with low pH. Heterodimers containing native α subunits or analogs of αG22C and αV53C α subunits were disrupted at low pH, suggesting that they lack intersubunit disulfide bonds. However, only a small fraction of heterodimers containing analogs of the αQ5C, αQ27C, αP40C, αK51C, αL41C, αM71C, and αV76C α subunits were shown to be stabilized by intersubunit disulfide bonds (Table 2). For analogs that form little or no heterodimers, the cysteine within the α subunit analog is located at the subunit interface or is far from the Asp111 residue of the β subunit, which is the β The first residue in the carboxy-terminal extension of the subunit. Thus, the access of the cysteine residue at position 138 of the β subunit to the cysteine on the α subunit appears to be blocked. This phenomenon suggests that most of the disulfide cross-links between subunits are formed when the subunits assemble into heterodimers with structures similar to hCG. Only one of the tested alpha subunit analogs formed a heterodimer with neither the hCG beta subunit nor hCG beta-S138C, which contained a substituted cysteine at Tyr89. Although this tyrosine can be removed or substituted into other residues except cysteine, and does not destroy the structure of the heterodimer (Pierce, J.G. & Parsons, T.F. (1981) Annu.Rev.Bioche m.50 , 465-495), is not necessary for the folding of the α subunit, and replacing the tyrosine with cysteine may still destroy the folding structure of the α subunit (Chem, F., Wang, Y. & Puett, D.( 1992) Mol. Endocrinol. 6, 914-919).

Table 2. The indicated α-subunit constructs and hCGβ-S138C used α subunit analogs Heterodimer (ng/50μl) Cross-linking (%) ^a Seat belt fastening (B11/B110) ^b natural alpha 26.2±0.8 -6.5±2.6 not calculated αQ5C 14.3±1.3 17.5±9.9 0.98 αG22C 9.1±0.4 -3.1±1.0 not calculated αQ27C 3.3±0.0 21.1±4.2 1.03 αR35C 6.0±0.0 98.4±4.9 0.97 αY37C 5.4±1.2 80.2±0.5 0.18 αP38C 0.3±0.04 Not tested not calculated αP40C 2.1±0.04 52.8±4.3 0.64 αL41C 3.2±0.04 37.2±0.6 0.49 αR42C 22.1±5.3 67.7±5.3 0.48 αS43C 29.7±1.4 81.3±1.9 0.60 αK44C 4.0±0.1 55.4±1.3 0.84 αK45C 5.8±0.1 82.9±6.2 0.85 αT46C 21.4±0.4 76.8±0.6 1.14 αM47C 4.9±0.8 72.0±2.0 0.82 αL48C 14.3±0.9 88.5±2.1 0.86 αV49C 6.0±0.5 70.5±6.1 0.76 αQ50C 0.8±0.1 66.7±7.4 0.82 αK51C 4.1±1.2 27.5±1.4 0.86 αN52C 6.3±0.2 70.6±3.9 0.88 αV53C 6.1±0.4 4.3±2.9 1.01 αS64C 4.4±0.3 82.5±8.9 0.85 αM71C 6.2±0.3 14.1±3.2 not calculated αV76C 4.3±0.0 41.9±4.9 1.10 αT86C 7.6±0.8 88.4±9.3 0.66 αY88C 7.4±0.3 83.0±6.2 not calculated αY89C not detectable no inspection not calculated αH90C 7.2±1.3 79.2±1.0 0.86 αK91C 9.9±5.7 69.4±3.3 0.91 αS92C 15.1±0.8 140.7±14.4 0.98

a) This value is calculated as the percentage of substance remaining in the sample after acid pH treatment as described in the text.

b) This value is the ratio of the activity measured with A133 and ¹²⁵ I-B111 in the sandwich assay to the activity measured with A113 and ¹²⁵ I-B110 after treatment with low pH. Any B111 binding is detected, indicating that the seat belt is fastened. The lower observed values in some cases may reflect the influence of the carboxyl terminus of the β subunit on the ability of B111 to interact with cross-linked heterodimers.

Numerous cysteine substitutions in the second loop of the alpha subunit residues had little effect on the receptor binding and signaling activity of hCG (Table 3, Figure 2). Replace αMet47 (Table 3, Figure 2) and αLys51 with cysteine (Einstein, M., Lin, W., Macdonald, G.J. & Moyle, W.R (2001) Exp.Biol.Med.226, 581-590 .) The α subunit residues reduced the activity of the heterodimer relative to that of hCG in binding and signaling assays (Table 3). However, the activity of analogues in which αLys51 is replaced by alanine is greatly reduced compared with hCG (Einstein, M., Lin, W., Macdonald, G.J. & Moyle, W.R. (2001) Exp.Biol.Med.226, 581-590. ), the activity of the analog hCG-αM47A, in which αMet47 was replaced by alanine, was almost comparable to that of hCG in both assays (Table 3). It is suggested that the presence of 47th methionine in the α subunit residue is not necessary to maintain hCG activity. The specific role of αLys51 in receptor interactions remains to be determined. The heterodimers in which the 51st residue of the α subunit is cross-linked with the 99th residue of the β subunit by a disulfide bond are more potent than those in which αLys51 is replaced by cysteine or alanine. Strong activity, based on this finding that substitution of the side chain of αLys51 has the potential to alter the conformation of the heterodimer.

Table 3. Effects of mutations in the second ring of the α subunit and the carboxyl terminal on the activity of heterodimer luteinizing hormone α subunit analogs Potency of native β subunit analogs (percent hCG, 95% CL) Potency of acid-stabilized hCGβ-S138C analogs (percent hCG, 95% CL) receptor binding ^a signaling ^a receptor binding ^a signaling ^a αR42C 113 (97-132) 103 (85-124) 327 (305-351) 332 (287-385) αS43C 35(31-39) 74(50-110) 17 (15-20) 43 (37-49) αK44C 66(60-72) 97(75-124) 250(228-274) 191 (148-247) αK44A 84 (71-101) Not transfected Not transfected αK44E, K45Q 58(54-61) 42(32-55) Not transfected Not transfected αK45C 93 (87-100) 91 (71-117) 223 (212-257) 168 (129-217) αT46C 38(35-42) 56(43-73) 50(44-57) 52(43-64) αM47C 8.7(7.4-10) 23(16-32) 4.1(2.9-5.9) 2.4(2.1-2.7) αM47A 83(71-97) Not transfected Not transfected αL48C 60(54-68) 147 (96-226) 66(60-72) 99 (81-120) αV49C 70(64-77) 133 (102-174) 71(65-76) 99 (84-119) αQ50C 81(76-85) 216 (163-285) 308(241-395) αK51C not detected ^* not detected ^* αN52C did not do did not do 61(57-65) αV53C 149 (137-162) did not do did not do αS64C did not do did not do 89 (78-101) αT86C 95 (84-106) 444 (354-555) 2.2 (Intermediate) 0.18(0.16-0.21) αY88C 141 (128-156) 74 (64-84) 0.05 (intermediate) 0.12 (0.09-0.16) αY89C did not do did not do did not do did not do αH90C 30(26-34) 65(48-89) 3.1(1.8-5.4) 1.0(0.96-1.1) αK91C 41(38-45) 56 (46-68) 2.2(1.1-4.4) not detectable αK91E 4.3(3.4-5.5) 1.1(0.1-2.2) Not transfected Not transfected αK91M 68 (36-144) 66(45-140) Not transfected Not transfected αS92C 100 (91-110) 38 (31-47) 28(23-34) 13 (12-14)

a) Analogue concentrations determined based on the sandwich immunoassay. These values were calculated from the IC50 values summarized for the experiments. Several analogues were not tested because the amount produced by the transfected COS-7 cells was too small. As shown in Table 1, α/hCGβ-S138C did not obtain any stable heterodimer after acid treatment.

It has been reported that changing αLys44 in the second ring of the α subunit to alanine reduces hCG activity by 100 times or more (Xia, H., Chen, F. & Puett, D. (1994) Endocrinol.134 , 1768-1770). However, we found that replacing αLys44 and several residues in the α subunit with cysteine had much less effect on hCG activity than expected (Table 3). In the course of partly uncorrelated studies testing predictions for the charge at the top of the second ring of the alpha subunit following subunit association (Slaughter, S., Wang, Y.H., Myers, R.V. & Moyle, W.R. (1995) Mol. Cell. Endocrinol.112,21-25), prepare the 44th and the 45th lysine respectively with the analog (hCG-αK44E, K45Q) (Fig. 15, SEQ ID NO:52) that glutamate and glutamine replace . Unexpectedly, this analog had the same activity as hCG-αK44A (Figure 15, SEQ ID NO:51) and hCG-αK44R (Figure 15, SEQ ID NO:53) in both assays, where the latter αLys44 of both has been replaced by alanine and arginine (Table 3, Figure 19). The high activity of the latter analogs could be predicted by the fact that the equine α-subunit contains the same substitution (Piece, J.G. & Parsons, T.F. (1981) Annu. Rev. Biochem. 50, 465-495). These studies are consistent with the results obtained by replacing these residues with cysteine, contradicting earlier findings (Xia, H., Chen, F. & Puett, D. (1994) Endocrinol. 134, 1768-1770), It is suggested that none of the highly conserved positively charged tyrosine residues on the small helix in the second loop of the α subunit is necessary for the LHR interaction.

Removal of βCys26

It has been found that if the carboxy-terminus of the seat belt is attached to βCys110 of the β subunit core, making it difficult to form a disulfide bond with residue 26 of the β subunit, this residue can interact with many of the α subunit analogs. Cysteine forms cross-links (Xing, Y., Lin, W., Jiang, M., Myers, R.V., Cao, D., Bernard, M.P. & Moyle, W.R. Other way folded chorionic gonadotropin analogs : Implications for hormone folding and biological activity. Journal of Biologicalchemistry.50, 46953-46960, 2001.). This finding changed the position of the seat belt and eliminated the phenotype of antibody B111. To see if the safety belt of the cross-linked analogs is linked to βCys26, the ability of these analogs to be recognized by antibody B111 and antibody B110, which recognize different β-subunit phenotypes, was compared. As shown in Table 2, each of the cross-linked heterodimers was recognized by B111, although not necessarily equally by B110, suggesting that the safety zone is linked to βCys26 in the same pattern as in hCG. Although we cannot rule out the possibility that βCys110 is linked to a cysteine introduced in the α subunit of some analogues, this possibility is almost non-existent. There are two reasons. First, the beta subunits used in these studies all contained a cysteine at residue 26. Deletion of cysteine at residue 26 is necessary for cross-linking of the seat belt with the cysteine introduced in the α subunit, regardless of its position (Xing, Y., Lin, W., Jiang , M., Myers, R.V., Cao, D., Bernard, M.P. & Moyle, W.R. Other folded analogs of chorionic gonadotropin: hints for hormone folding and biological activity. Journal of Biological chemistry.2001.). Second, the position of the cysteine least likely to be recognized by B111 in the alpha subunit of the cross-linked analog is close to the binding site of B111 (Fig. 1). These suggest that crosslinking stabilizes the position of the carboxyl terminus of the β subunit of these analogs in a position that mediates B111 access to the heterodimer.

α subunit carboxy terminus

The carboxyl terminus of the α-subunit is thought to be essential for LHR interaction (Piece, J.G. & Parsons, T.F. (1981) Annu. Rev. Biochem. 50, 465-495; Yen, S.S.C., Llerena, O., Little, B. & Pearson , O.H. (1968) J. Clin. Endocrinol. Metab. 28, 1763-1767). The presence of the carboxy-terminal cysteine of the α-subunit also resulted in a slight decrease in heterodimer activity (Table 3, Figure 3), a phenomenon consistent with the putative role of this portion of the hormone for receptor contact (Piece, J.G. & Parsons, T.F. (1981) Annu. Rev. Biochem. 50, 465-495; Chen, F., Wang, Y. & Puett, D. (1992) Mol. Endocrinol. 6, 914-919). However, the activity of these analogs is much stronger than that of analogs lacking the five carboxy-terminal α subunit residues (Piece, J.G. & Parsons, T.F. (1981) Annu. Rev. Biochem. 50, 465-495), suggesting that this region Any single residue within is not required for hormonal activity, and each residue makes only a small contribution to the total energy of hCG binding to LHR. Based on the putative contact of αLys91 with aspartic acid in the transmembrane domain (Yoo, J., Ji, I. & Ji, T.H. (1991) J.Biol.Chem.266, 17741-17743), αLys91 plays an important role in signaling is essential in (Ji, I., Zeng, H. & Ji, T.H. (1993) J.Biol.Chem.268, 22971-22974), thus the heterodimer containing αK91C retains most of the energy of hCG (Figure 3) is surprising. The finding that hCG-[alpha]K91C retains substantial potency is inconsistent with this hypothesis. Therefore, hCG-αK91E and hCG-αK91M were prepared and their activities were examined. These analogs are reported to have very low potency (Yoo, J., Ji, I. & Ji, T.H. (1991) J. Biol. Chem. 266, 17741-17743). As shown in Figure 17, both analogs had substantial potency, although the analog containing glutamate interacted with LHR with a 10-fold lower affinity than hCG. This observation is consistent with that obtained with the conversion of αLys91 to cysteine (Table 3, Figure 3), suggesting that this lysine residue is not very important for signaling as reported (Yoo, J., Ji, I. & Ji, T.H. (1991) J. Biol. Chem. 266, 17741-17743).

Carboxy terminus of β subunit and second loop of α subunit

Many of the acid-stable analogs in which the carboxyl terminus of the β subunit was cross-linked with residues in the second loop of the α subunit had appreciable activity in receptor binding and signaling assays (Table 3, Figure 4). These analogs included those linked to residues 35, 37, 40, 44, 42, 50 and 52 of the second loop of the alpha subunit (Table 3). The carboxyl terminal of the β subunit is connected to the 47th position on the second ring of the α subunit, so that the receptor binding activity is almost completely lost, and the coupling with the 43rd and 46th positions reduces the activity of the heterodimer by half. In the most active analogues, the side chains of the residues in the second loop of the α subunit protrude toward the first and third loops of the β subunit in almost all of the analogues, suggesting that facing the first and third loops of the β subunit The α subunit of the first ring and the third ring The surface of the second ring is not in contact with the LHR. The side chains of the 43rd and 46th residues of the α subunit protrude toward the safety belt, and the side chain of the 47th residue faces the small loop of the safety belt formed by the disulfide bond between Cys93 and Cys100. Therefore, the loss of activity caused by the cross-linking of the carboxyl terminus of the β subunit to these sites may disrupt the interaction between these parts of the second loop of the α subunit and the receptor, or reduce the ability of the hormone to interact with the receptor. The conformation of the second loop of the α subunit is changed in a manner.

The length of the carboxy-terminal end of the β-subunit suggests that residue 138 can pass over the first and third loops to connect to residues on the second loop (Figure 1). Thus, the carboxy-terminal portion of any of these analogs may not occupy the groove space between the second ring of the alpha subunit and the first and third rings of the beta subunit. In order to study the activity of analogues whose carboxy-terminal part of the β-subunit occupies this groove, analogues with residues 116-135 and residues 121-135 of the β-subunit deleted were prepared. The beta carboxyl termini of these analogs are too short to form a crosslink with residues on the second loop of the alpha subunit without traversing the intersubunit groove. These analogs were substantially active in both LHR and signaling assays (Fig. 4), providing further support for the notion that this part of the second loop of the alpha subunit does not make contact with LHR.

The first and third rings of β subunit and α subunit

To see whether residue 138 of the β subunit can cross-link with other parts of the α subunit core, hCG-βS138C was mixed with several α subunits with cysteine-substituted residues in the first or third loop. expressed together with analogues. Except for heterodimers containing a substituted cysteine at position 64 of αSer, only a small fraction of the resulting heterodimers were cross-linked because of their low stability at acidic pH (Table 2) . Many of these heterodimeric α-subunit analogs have cysteines located much farther from the carboxy-terminus of the seatbelt than analogs that participate in subunit cross-linking, possibly beyond the β-subunit. The range that the carboxy-terminal region of the carboxyl group can effectively contact (Figure 1). In receptor binding and signaling activity assays, the αS64C/hCG-βS138C heterodimer had appreciable activity (Table 3), indicating that this α subunit residue does not make the necessary contacts with LHR.

Cross-linking of the carboxyl-terminal residues of the β-subunit to the carboxyl-terminal residues of the α-subunit had a greater effect on the interaction of the heterodimer with the LHR than did cysteine substitution in either subunit (Fig. 5 ). It is suggested that the carboxy-terminus of the α-subunit is close to the contact surface of the LH receptor, as assumed by studies done more than 25 years ago (Piece, J.G. & Parsons, T.F. (1981) Annu. Rev. Biochem. 50, 465-495). However, attachment of the carboxyl terminus of the beta subunit to this region may alter the structure of the heterodimer or cause this end of the beta subunit to move closer to other parts of the hormone that make contact with the receptor.

protein nodule-hCG-beta-lactamase

These studies provide substantial support for the notion that most residues on the second loop of the α subunit are not involved in essential LH receptor contacts. However, these studies do not rule out the possibility that this part of the hormone contacts close to the receptor. To clarify this problem, hCGβ-subunit analogues were prepared by linking a globular protein similar in size to hCG, β-lactamase, to a specific position on the α-subunit. These analogs were prepared by fusing β-lactamase to residues 140 and 145 of hCG-βS138C to generate hCG-βS138C-βLA140 and hCG-βS138C-βLA145, respectively. The latter analog contains a "spacer" comprising seven residues between the cysteine cross-linked to the alpha subunit and the cysteine cross-linked to the amino terminus of the beta-lactamase. Coexpression of these β subunit analogs with the α subunit analogs αT46C, αL48C, αS64C and αS92C resulted in the formation of acid stable heterodimers (Figure 6). This result shows that the presence of β-lactamase does not prevent the attachment of residue 138 of the β subunit to the α subunit.

These hCG-β-lactamase analogs were much less active than the corresponding analogs without β-lactamase (Figure 6).

Contrary to previous studies designed to identify parts of the α subunit not involved in key receptor contacts (Xing, Y., Lin, W., Jiang, M., Myers, R.V., Cao, D., Bernard, M.P. & Moyle, W.R. Other-folded chorionic gonadotropin analogs: Implications for hormone folding and biological activity. Journal of Biological chemistry. 2001.) Taken together, these results extend the current understanding of surfaces not in contact with the LHR and suggest that α The groove between the second loop of the subunit and the first and third loops of the β subunit is not involved in the necessary LH receptor contacts that were critically required in a previous model (Moyle, W.R., Campbell , R.K., Rao, S.N.V., Ayad, N.G., Bernard, M.P., Han, Y. & Wang, Y. (1995) J. Biol. Chem. 270, 20020-20031.). Indeed, based on the ability of monoclonal antibodies to recognize hCG and its analogs bound to LHR on the cell surface (Wang, Y.H., Bernard, M.P. & Moyle, W.R. (2000) Mol. Cell. Endocrinol. 170, 67-77), and Binding ability of hCG/hFSH and hCG/hTSH chimeras to LHR [Campbell, R.K., Dean Emig, D.M. & Moyle, W.R. (1991) Proc.Natl.Acad.Sci. (USA) 88,760-764; Campbell, R.K. , Bergert, E.R., Wang, Y., Morris, J.C. & Moyle, W.R. (1997) Nature Biotech.15, 439-443; Grossmann, M., Szkudlinski, M.W., Wong, R., Dias, J.A., Ji, T.H. & Weintraub, B.D. (1997) J.Biol.Chem.272, 15532-15540; Moyle, W.R., Campbell, R.K., Myers, R.V., Bernard, M.P., Han, Y. & Wang, X. (1994) Nature 368, 251 -255], few hCG-specific residues appear to be involved in essential LHR contacts. Residues that have a large effect on hCG activity lie within the safety band, but individual changes of these residues do not alter the hormone-receptor interaction several-fold (Han, Y., Bernard, M.P. & Moyle , W.R. (1996) Mol. Cell. Endocrinol. 124, 151-161). Two distant sites on the receptor may affect hCG-LHR interaction (Bernard, M.P., Myers, R.V. & Moyle, W.R. (1998) Biochem.J.335, 611-617), while mammalian luteinizing hormone and The human LHR interaction is very sensitive to small conformational changes in the hormone. Based on these observations, we suggest that the interaction of glycoprotein hormones with their receptors is not dominated by relatively few contacts, just as that of growth hormone and its receptors [Clackson, T. & Wells, J.A. (1995) Science 267, 383-386; Wells, J.A. (1996) Proc. Natl. Acad. Sci. (USA) 83, 1-6].

Example 2 - Investigating the protein nodule of hFSH docking to the FSH receptor

The cDNA encoding the beta subunit of hFSH corresponding to the translated portion of the mRNA was obtained from Christie Kelton, Ares Advanced Technology, 280 Pind Street, Randolph MA, a division of Serono. The amino acid sequence of the β subunit without the leader peptide is shown in ( FIG. 8 , SEQ ID NO: 40). hFSH was improved by PCR and cassette mutagenesis to generate a construct named FC1-108β that encodes residues 1-108 of hFSH and residues 115-145 of hCG in tandem (Figure 8, SEQ ID NO :41). The amino acid sequence of FC1-108β without leader peptide found in hFSH β subunit is also shown in FIG. 8 . Replace the ShoI-Apal fragment of the construct encoding hCG-S138Cβ with the ShoI-Apal fragment of FC1-108β to prepare the FC1-108β analog with the Ser132 residue converted into Cys, thereby generating FC1-108, S132Cβ (Fig. 8 , SEQ ID NO: 42). Several analogues of FC1-108β, S132Cβ and α subunit shown in FIG. 7 were expressed in COS-7 cells by the method described in Example 1. The amount of heterodimer secreted by the cells into the medium was determined using antibodies A113 and ¹²⁵ I-B603. The former antibody is an antibody against α subunit, and the latter antibody is a monoclonal antibody combined with hFSH β subunit. Purified hFSH was used as standard. After acid treatment to dissociate the non-crosslinked heterodimer, the assay was repeated with A113 ^and125I -B603 as described above to determine the amount of crosslinked material in the sample. The resulting cross-linked analogs were tested for their ability to stimulate the accumulation of cyclized AMP in CHO cells expressing the FSH receptor.

As shown in Figure 11, the nodule located at residue 35 of the alpha subunit only slightly decreased the activity of this FSH analogue. Attachment of the nodule to residues 92, 64, 48, 46, 42, 90, 43, 88 and 86 resulted in a progressive loss of activity of the analog. It shows that the 35th residue of α subunit is not close to the FSH receptor contact surface, while several other residues seem to be close to the receptor contact surface. Unlike the interaction of hCG with the LH receptor, the presence of nodules at residues 42 and 43 exerted a stronger inhibitory effect on the binding of this FSH analogue to its receptor. This suggests that when FSH and hCG interact with their receptors separately, the surface portion of the second loop of the alpha subunit is closer to the FSH receptor than to the LH receptor. The result that the nodules are linked to residue 86 of the alpha subunits of both receptors indicates that this part of both ligands is close to the receptor interface.

Example 3——Study on the protein nodules of bifunctional hCG/hFSH chimera docking to LH and FSH receptors

The cDNA sequence encoding the chimera was prepared by conventional methods, wherein the codons of amino acids 101-114 of hCG were replaced by the corresponding part of hFSH β subunit. The amino acid sequence of the chimera was named CFC101-114β, and Figure 8 (SEQ ID NO: 38) shows the sequence except the leader peptide residue. Replace the ShoI-ApaI fragment of the construct encoding hCG-S138Cβ with the ShoI-ApaI fragment of CFC101-114β to prepare the CFC101-114β analogue in which the Ser138 residue is converted into Cys, thereby generating CFC101-114β, S132C (Fig. 8 , SEQ ID NO: 39). Several analogues of CFC101-114β, S132C and α subunit shown in FIG. 7 were expressed in COS-7 cells by the method described in Example 1. The amount of heterodimer secreted by the cells into the medium was determined using antibodies A113 ^and125I -B110. The former antibody is an antibody against α subunit, and the latter antibody is a monoclonal antibody combined with hCG β subunit. Purified hCG was used as standard. After acid treatment to dissociate the non-crosslinked heterodimer, the assay was repeated with A113 ^and125I -B110 as described above to determine the amount of crosslinked material in the sample. The resulting cross-linked analogs were examined for their ability to inhibit the binding of ¹²⁵ I-hCG to CHO cells expressing LH receptors, and the ability to inhibit the binding of ¹²⁵ I-hFSH to CHO cells expressing FSH receptors ( FIG. 12 ). At the same time, its ability to stimulate the accumulation of cyclic AMP in FSH receptor-expressing CHO cells and FSH receptor-expressing CHO cells was also examined.

As shown in Figure 12, the nodule located at residue 35 of the α subunit prevents radiolabeling of hCG or radiolabeling of hFSH and LH in this chimera compared to bifunctional chimeras lacking most of the carboxyl terminus of the hCG β subunit, respectively. or the ability to bind to FSH receptors will not interfere. However, the presence of a nodule at residue 37 of the α subunit leads to dramatically different results. This nodule had little effect on the chimera's ability to bind to the LH receptor, but almost completely abolished its ability to bind to the FSH receptor. The presence of nodules at the 43rd and 46th positions of the α subunit decreased the binding ability of the chimera to both receptors at the same time. The presence of nodules at positions 48 and 52 of the alpha subunit had much less effect on the ability of the chimeras to bind either receptor. As observed in Examples 1 and 2, binding of the chimera to both receptors was severely inhibited by the presence of the nodule at position 86 of the alpha subunit. Both this portion of the hormone and the interface between the chimera and the two receptors were shown to be in close proximity. Similar results were observed for the nodule at position 91 of the α subunit.

As shown in Figures 13 and 14, the presence of nodules reduced the activity of most chimeras in LH receptor and FSH receptor signaling assays. However, the degree of activity reduction varies with the receptors tested by the test substance. For example, the presence of a nodule at position 37 of the α subunit inhibited FSH receptor-induced cyclic AMP accumulation much more strongly than LH receptor-mediated cyclic AMP accumulation. Altogether, the relative impact of nodules on the chimera's ability to elicit cellular responses was similar to its ability to affect receptor interactions. Thus, even though LH and FSH have similar structures, and even though their receptors are very similar, these ligands do not interact with the receptors in the same way. This example demonstrates the ability to use these probes of the invention to identify protein-protein interactions.

Example 4: Nodules containing β-lactamase

Analogs containing the hCGβ, S138Cβ subunit have a relatively small nodule consisting of residues surrounding Ser138. These nodules are large enough to measure the relatively small distance between the ligand and its receptor. However, these nodules are still too small to be used to detect the proximity of residues that are more distant from the receptor. This limitation can be circumvented by adding β-lactamase to make the probe larger. [beta]-lactamase was chosen as a probe because its crystal structure is known and its carboxy- and amino-termini are located on the surface of the protein. This is useful for generating fusion proteins at either end of the hormone. Another advantage of selecting β-lactamase is that it is a high conversion factor, enzyme that cleaves fluorescent substrates (Zlokarnik, G., Negulescu, P.A., Knapp, T.E., Mere, L, Burres, N., Feng , L., Whitney, M., Roemer, K. & Tsien, R.Y. (1998) Science 279,84-88), this point is useful when detecting fusion proteins. According to this, β-lactamase has been used as an effective reporter molecule, although the use is quite different from that envisioned here (Moore, J.T., Davis, S.T. & Dev, I.K. (1997) Anal.Biochem.247, 203- 209). β-lactamases can also be inhibited by proteins that bind to them (Strynadka, N.C., Jensen, S.E., Johns, K., Blanchard, H., Page, M., Matagne, A., Frere, J.M. & James, M.N. (1994) Nature 368, 657-660.). This makes it possible to further enlarge the beta-lactamase nodules by simply adding arrestin.

Preparation of two hCGβ,S138C derivatives containing β-lactamase fusion protein. One of them was named hCGβ, S138C-βLA(long) (SEQ ID NO: 44), or long (LONG) for short. As shown in Figure 15, it contains β-lactamase fused to the carboxyl terminus of hCGβ,S138C. The protein was prepared by PCR mutagenesis using pUC18 as a template. Another probe was designated hCGβ, S138C-βLA (short) (SEQ ID NO: 43), or simply short (SHORT). As shown in Figure 15, it contains the β-lactamase fused to the truncated hCGβ, S138C. Short proteins have only one amino acid between the coupled cysteine and the start position of the β-lactamase. Clearly, different versions of these proteins can be made by inserting different numbers of residues between the coupled cysteine and the β-lactamase. At the same time, it should be recognized that those skilled in the field of molecular modeling and molecular biology can design and prepare a β-lactamase analog whose surface residue is replaced by cysteine. This cysteine can serve as a coupling cysteine, which allows the [beta]-lactamase nub to be anchored to the target cysteine with a more rigid linkage than with the tail.

Generates five distinct stable heterodimers containing long and short β subunits. These dimers were prepared to determine the proximity of residues 46, 48, 52, 64 and 92 of the alpha subunit relative to the LH receptor. As shown in Figures 16 and 17, none of these analogs had comparable activity to hCG. Heterodimers of long and short β subunit probes coupled to residue 52 of α subunit [i.e. αN52C+hCGβ, S138C-βLA(long) and N52C+hCGβ, S138C-βLA(short) respectively ] retained most of the activity of hCG in signaling assays. Changing the α-subunit Asn52 residue to cysteine disrupts the glycosylation signal, resulting in loss of the oligosaccharide on the second loop of the α-subunit. Loss of this oligosaccharide from hCG resulted in a nearly 60% drop in its potency (Figure 18). Thus, attachment of a β-lactamase knot at this site can result in loss of the oligosaccharide on the second loop, thereby losing its potency. Both these long and short analogs have relatively high activity, indicating that the α subunit residues are not close to the receptor interface. Given the high potency of both analogs, these results suggest a role for oligosaccharides in hCG-mediated signaling. The activity of the long and short analogues suggests that the role of the oligosaccharide on the second loop of the α subunit is to disrupt the position of the subunits within hCG, which is independent of specific contacts between the oligosaccharide and either subunit.

The activities of the long and short analogues were still maintained, indicating that the 48th and 64th residues of the α subunit were closer to the receptor than those determined by the activities of hCGβS18C+αL48C and hCGβS138C+αS64C. This finding has important implications for models of the hCG-LH receptor complex. It also confirms the earlier finding that residues 46 and 92 of the α subunit are close to the receptor surface.

Example 5: The N-terminus of the α-subunit has a cysteine-coupled nodule

The coupled cysteine does not have to be located at the carboxy-terminal of the protein, we designed an analog with the coupled cysteine located at the amino-terminal of the protein. The amino acid sequence of such an analog (β101-145,α) is shown below. The preparation of this analog includes deleting the residues of the 3rd to 100th β subunits of hCG, and fusing the α subunits to the ends of the obtained β subunits by conventional PCR and cassette mutagenesis methods. This analog contains a free cysteine at residue 12 that can serve as a coupling cysteine.

10 20 30 40 50 60

β101-145, α (SEQ ID NO: 54)

skggpkdhpltcddprfqdsssskapppslpspsrlpgpsdtpilpqapdvqdcpectlq-

70 80 90 100 110 120

enpffsqpgapllqcmgccfsrayptplrskktmlvqknvtsestccvaksynrvtvmgg-

130 140

fkvenhtachcstcyyhks

Example 6 - Method for generating site-specific protein nodules

Select the protein to which the nodule will be attached. Then, identify the specific sites on the protein to be tagged. Constructs expressing the protein are prepared by mutagenesis methods known in the art by replacing the native residue at the second protein-specific marker with cysteine. In addition, the protein encoded in the construct also includes a tail containing a cysteine attached to one end of the protein and a knot attached to the end of the tail. The construct is then inserted into expression cells, resulting in the protein with the nub attached at the specific site.

Adding nodules to proteins involves introducing cysteines on the surface where the nodules need to be located, and introducing cysteines at the amino-terminal or carboxy-terminal positions of the protein so that the cysteines at the end of the protein can be aligned with The cysteines that have been added to the surface form disulfide bonds. The formation of a disulfide bond between these two cysteines will stabilize the cysteine-containing protein on the surface generating nodules at this site (see Figure 9).

In order to generate a nodule, the cysteine-containing protein terminus needs to be long enough that its cysteine can form a disulfide bond with a cysteine at the site that will contain the nodule. This may necessitate the addition of a tail such as the carboxyl terminus of the hCG β subunit, as is the case with hFSH plus protein knobs. The composition of the tail can vary widely and is not limited to the carboxyl terminus of the hCG β subunit. The main requirement for the tail is that it be long enough to be able to interact with the cysteine on the surface of the protein that defines the position of the nodule, while being free of residues that would prevent its access to this site. These include residues characteristic of transmembrane domains, as well as residues that create binding sites for other proteins, or parts of them, that are far from cysteines on the surface of the protein where the nodule is located.

Residues on either side of a cysteine at the amino- or carboxyl-terminus of a protein can form nodules (see Figure 9). Generally, the more residues at this site, the larger the nodule. The smallest nodules may contain only one cysteine (Fig. 10A). The generation of such nodules involves the introduction of a cysteine at the amino-terminal or carboxy-terminal end of the linker, and the introduction of a cleavage site immediately adjacent to the cysteine (see Figure 10A). The cleavage site should contain an amino acid sequence that is not found in other parts of the protein and can be hydrolyzed by proteases. There are at least three ways a nodule can get bigger. The first way is to fuse a protein at the end to the protein that builds the nodule (see Figure 10B). β-lactamase was fused to the protein terminus as previously described. However, beta-lactamases are not the only proteins suitable for this purpose. The convenience of choosing β-lactamase as a probe is that its crystal structure shows that its amino terminus is on the surface, which is convenient for the construction of fusion proteins. The second way is to cleavage the linker with a protease, reducing the size of the linker and its effect on the nodule. Third, by increasing or decreasing the number of amino acids, the distance between the coupled cysteine and the fusion protein probe is changed. As seen in the hCG example, the movement of the nodule is restricted by shortened distances, and the activity of the hormone decreases more severely with shorter distances. This keeps the nodule close to the interface between the hormone and the LH receptor.

As evident from Figure 10, the coupled cysteines do not have to be located at the tail. Introducing a cysteine on the surface of the fusion protein that is part of the nodule, where it can form a disulfide bond with the target cysteine on the protein, will allow the fusion protein to attach directly to the surface of the protein to be probed (see Figure 10C ). This method can be used to control the orientation of fusion proteins that are part of the nodule. When proteases are used as fusion proteins, the introduction of cysteines on surfaces remote from the active site keeps the active site of the fusion protein away from the surface to which the nodule will be attached.

Example 7: Using truncated tails as probes

Adding nodules to proteins enables the determination of the distance between two protein surfaces. As shown, the second loop of the alpha subunit does not substantially contact the receptor. The positioning of the coupling cysteine at amino acid 138 of the hCG β subunit places relatively few constraints on the position that can be occupied by residues 111-137 of the β subunit, of which residues 111-137 would be on the nodule The coupled cysteines are linked to the core of the hCG β subunit. The tail itself can also be used to probe the surface of a protein, and there have been several examples of constraining its position to a specific location on the molecule. Examples of these include capping part of the protein's active site, making it inactive until the tail is cleaved away by a protease.

In the hCG-LH receptor interaction study, in order to test the assertion that the second loop of the α subunit forms a critical receptor contact with the groove between the first and third loops of the β subunit, the tail Confining to this ditch would be ideal. Acting on the aforementioned tail will allow it to pass through this part of the hormone, but not necessarily force it to be positioned in this position. Due to its length, the tail may pass over the convexity of the first and third loops of the β subunit so that the coupled cysteine of the knob can be replaced by a different cysteine on the second loop of the α subunit touch. Therefore, when targeting residues 42, 46, or 48 on the second loop of the α subunit to replace cysteine, we truncated the tail to force it to traverse the second loop of the α subunit and the β The groove between the first and third rings of a subunit. Tail mimetics hCGβ, δ116-135, S138C (SEQ ID NO: 45) and hCGβ, δ121-135, S138C (SEQ ID NO: 46) (FIG. 15) were too short in length to make the distance between the nodule and the target protein Disulfide bonds are formed unless the tail traverses the groove between the second loop of the α subunit and the first and third loops of the β subunit. As can be seen from the results in Figure 23 and Figure 24, the heterodimer containing αT46C and hCGβ, δ116-135, S138C or hCGβ, δ121-135, S138C has substantial receptor binding activity. The heterodimer containing αL48C and hCGβ, δ121-135, S138C also has considerable receptor binding activity. Fewer heterodimers containing αL48C and hCGβ, δ121-135, S138C, and acid-stable heterodimers were formed, suggesting that the shortest tail tested may have the ability to make the nodule-coupled cysteine contact with the target cysteine on the protein of sufficient length. Since the nodule-coupled cysteine makes contact with the target cysteine at residue 42 of the α subunit, the tail of the analog does not necessarily traverse the second loop of the α subunit and the first loop of the β subunit and the For the groove between the third ring, choose this analog as a positive control.

Taken together, these observations suggest that this groove is not involved in essential receptor contacts. It also shows how to manipulate the position of the tail to bring it closer to a specific part of the protein. This allows the tail to be used to mask specific sites, a property which, in combination with cleavage sites already introduced into the tail, would be very useful in the preparation of latent proteases, toxins, or other useful analogs. Tails that shield protease or toxin action sites can be used to prepare and use reagents that can enter cells, and these reagents can be cleaved by endogenous or other hydrolytic enzymes after entering cells to activate the toxin. These reagents will be very useful in the treatment of tumors or other diseases.

Example 8. Use of Probes Linked to β Subunits

The small loop in the seat belt is said to play a role in hCG's biological activity, and to investigate this possibility it was necessary to attach a nodule to this part of the β subunit. [alpha]-subunit analogs containing the carboxy-terminal residues of the hCG [beta]-subunit were prepared. Although attachment of a portion of the carboxyl terminus of the beta subunit to the carboxy terminus of the alpha subunit has been shown to reduce the activity of the heterodimer by 50-fold or more, using the entire beta subunit sequence does not have this effect. Analog containing the complete carboxy-terminal sequence of the hCG beta subunit linked to the alpha subunit, including the amino acid sequence Asp-Asp-Pro-Arg-Phe-Gln-Asp-Ser-Ser-Ser-Ser-Lys-Ala-Pro-Pro -Pro-Ser-Leu-Pro-Ser-Pro-Ser-Arg-Leu-Pro-Gly-Pro-Ser-Asp-Thr-Pro-Ile-Leu-Pro-Gln, in receptor binding and signal transduction assay its The activity is 50% or more of the activity of hCG. We found that this may be due to the existence of charged residues near the junction of the α-subunit-derived residues and β-subunit-derived residues. To attach the nodule to the β subunit residue of the safety belt small loop, a truncated hCG β subunit carboxy-terminus was attached to the end of the α subunit to generate hCG-αCTδ116/135, S138C (SEQ ID NO:57 ). Combine it with Arg94 (SEQ ID NO: 58), Arg95 (SEQ ID NO: 59), Ser96 (SEQ ID NO: 60), Thr97 (SEQ ID NO: 61), Thr98 (SEQ ID NO: 62) and Asp99 ( SEQ ID NO:63) Cysteine-substituted beta subunit analogs were co-expressed in COS-7 cells. From the data in Figures 26, 27, 28, 29, 30, 31 and Figure 32, it can be seen that these proteins all form acid-stable heterodimers. These studies reveal how this part of the β subunit interacts with the LH receptor (LHR )interaction. The presence of nodules at residues 95 and 99 abolished LHR interaction and biological activity. The effect of the nodule was much less when it was located at residue 96 or 97, as these analogs were essentially active in binding and signaling assays. The nodule further attenuated the receptor interaction when it was attached to residue 98, but not to the extent when attached to residues 95 and 99. It is suggested that the latter may be located close to the receptor interface.

The orientation of the side chain of residue 95 of the hCGβ subunit is opposite to that of residues 94 and 95, which may explain the fact that the junction at this position is more than that at residues 94 and 96. The fact that nodules are more able to suppress the hCG-LHR interaction. This point suggests that the surface of the seat belt near the side chain of Arg95 may be close to the receptor contact surface. To see if this side chain is in contact with the surface of the protein, analogs containing smaller knuckles were made. The construction methods of these analogs include replacing the 92nd serine (SEQ ID NO: 35) of the α subunit with cysteine, or connecting the tail containing Gly-Gly-Cys to the carboxyl terminus of the α subunit (SEQ ID NO :36). As can be seen from Figure 30 and Figure 32, smaller nodules have a much lower ability to interfere with heterodimer activity in the LHR assay. Thus, both acid-stable cross-linked heterodimers retained appreciable activity in these assays. It shows that the side chain of Arg95 residue of β subunit is very likely to be close to the receptor contact surface, but it is not necessary for receptor contact. At the same time, it also shows that the position of the carboxyl terminal of the α subunit after the interaction between the hormone and LHR is close to the small loop of the safety belt.

Since, like the oligosaccharide at Asn52 of the second loop of the α-subunit, the carboxy-terminus of the α-subunit is required for full glycoprotein hormone activity, how does the position of the carboxy-terminus of the α-subunit change in the heterodimer It would be very interesting to influence its biological activity. Preparation of α-subunits that cannot be glycosylated on the second ring of α-subunits due to the replacement of Asn52 with Asp, and which can add cysteine knots to residues 92, 94, 95, and 96 of β-subunits substance (SEQ ID NO: 65). Coexpression of this analog with β-subunit analogs containing substituted cysteines at residues 92, 94, 95, and 96 in COS-7 cells results in acid-stable cross-linked heterodimerization body. The activity of these analogs and the chimeric β-subunit analog (SEQ ID NO:66) containing a substituted cysteine at Arg96 indicates that the carboxy-terminus of the α-subunit can be linked to several sites in the β-subunit , indicating that its position is not properly secured (Figure 33). Furthermore, the low activity of the chimeric analogs suggests that the conformation of this part of the hormone has an effect on signaling.

The present invention is well adapted to practice, attaining the end results and advantages mentioned and inherent therein, as will be readily apparent to those skilled in the art. The methods and procedural steps and compositions described herein are representative of presently preferred embodiments, are illustrative, and are not intended to limit the scope of the invention. Those skilled in the art can make changes or use it for other purposes around the spirit of the present invention or the scope defined by the claims.

Various substitutions and modifications of the invention disclosed herein will be apparent to those skilled in the art that do not depart from the scope and spirit of this invention.

Patents or patent applications are hereby incorporated by reference in the same manner as each individual publication was specifically, individually indicated to be incorporated by reference in its entirety. The following are references that may be incorporated by reference.

references:

1. Lapthorn, A.J., Harris, D.C., Littlejohn, A., Lustbader, J.W., Canfield, R.E., Machin, K.J., Morgan, F.J. & Isaacs, N.W. (1994) Nature 369, 455-461.

2. Wu, H., Lustbader, J.W., Liu, Y., Canfield, R.E. & Hendrickson, W.A. (1994) Structure 2, 545-558.

3. Suganuma, N., Matzuk, M.M. & Boime, I. (1989) J.Biol.Chem.264, 19302-19307.

4. Wang, Y.H., Bernard, M.P. & Moyle, W.R. (2000) Mol. Cell. Endocrinol. 170, 67-77.

5. Segaloff, D.L. & Ascoli, M. (1993) Endocr. Rev.14, 324-347.

6. Kobe, B. & Deisenhofer, J. (1993) Nature 366, 751-756.

7. Braun, T., Schofield, P.R. & Sprengel, R. (1991) EMBO. J.10, 1885-1890.

8. Thomas, D., Rozell, T.G., Liu, X. & Segaloff, D.L. (1996) Mol. Endocrinol.10, 760-768.

9. Bernard, M.P., Myers, R.V. & Moyle, W.R. (1998) Biochem. J. 335, 611-617.

10. Cosowsky, L., Rao, S.N.V., Macdonald, G.J., Papkoff, H., Campbell, R.K. & Moyle, W.R. (1995) J.Biol.Chem.270, 20011-20019.

11. Moyle, W.R., Campbell, R.K., Rao, S.N.V., Ayad, N.G., Bernard, M.P., Han, Y. & Wang, Y. (1995) J.Biol.Chem.270, 20020-20031.

12. Myers, R.V., Wang, Y. & Moyle, W.R. (2000) Biochim. Biophys. Acta 1475, 390-394.

13. Jiang, X., Dreano, M., Buckler, D.R., Cheng, S., Ythier, A., Wu, H., Hendrickson, W.A., Tayar, N.E. & el Tayar, N. (1995) Structure 3, 1341 -1353.

14. Peng, K.C., Bousfield, G.R., Puett, D. & Ward, D.N. (1996) Journal of Protein Chemistry 15, 547-552.

15. Xia, H., Chen, F. & Puett, D. (1994) Endocrinol.134, 1768-1770.

16. Piece, J.G. & Parsons, T.F. (1981) Annu. Rev. Biochem. 50, 465-495.

17. Chen, F., Wang, Y. & Puett, D. (1992) Mol. Endocrinol. 6, 914-919.

18. Campbell, R.K., Dean Emig, D.M. & Moyle, W.R. (1991) Proc. Natl. Acad. Sci. (USA) 88, 760-764.

19. Campbell, R.K., Bergert, E.R., Wang, Y., Morris, J.C. & Moyle, W.R. (1997) Nature Biotech.15, 439-443.

20. Grossmann, M., Szkudlinski, M.W., Wong, R., Dias, J.A., Ji, T.H. & Weintraub, B.D. (1997) J. Biol. Chem. 272, 15532-15540.

21. Lindau-Shepard, B., Roth, K.E. & Dias, J.A. (1994) Endocrinol.135, 1235-1240.

22. Moyle, W.R., Matzuk, M.M., Campbell, R.K., Cogliani, E., Dean Emig, D.M., Krichevsky, A., Barnett, R.W. & Boime, I. (1990) J. Biol. Chem. 265, 8511- 8518.

23. Xing, Y., Lin, W., Jiang, M., Myers, R.V., Cao, D., Bernard, M.P. & Moyle, W.R. Alternatively folded choriogonadotropin analogs: implications for hormone folding and biological activity. Journal of Biological Chemi stry .2001.

24. Moyle, W.R., Ehrlich, P.H. & Canfield, R.E. (1982) Proc. Natl. Acad. Sci. (USA) 79, 2245-2249.

25. Moyle, W.R., Campbell, R.K., Myers, R.V., Bernard, M.P., Han, Y. & Wang, X. (1994) Nature 368, 251-255.

26. Matzuk, M.M. & Boime, I. (1988) J. Cell Biol. 106, 1049-1059.

27. Einstein, M., Liu, W., Macdonald, G.J. & Moyle, W.R. (2001) Exp. Biol. Med. 226, 581-590.

28. Slaughter, S., Wang, Y.H., Myers, R.V. & Moyle, W.R. (1995) Mol. Cell. Endocrinol. 112, 21-25.

29. Yen, S.S.C., Llerena, O., Little, B. & Pearson, O.H. (1968) J. Clin. Endocrinol. Metab. 28, 1763-1767.

30. Yoo, J., Ji, I. & Ji, T.H. (1991) J. Biol. Chem. 266, 17741-17743.

31. Ji, I., Zeng, H. & Ji, T.H. (1993) J. Biol. Chem. 268, 22971-22974.

32. Han, Y., Bernard, M.P. & Moyle, W.R. (1996) Mol. Cell. Endocrinol. 124, 151-161.

33. Clackson, T. & Wells, J.A. (1995) Science 267, 383-386.

34. Wells, J.A. (1996) Proc. Natl. Acad. Sci. (USA) 83, 1-6.

35. Zlokarnik, G., Negulescu, P.A., Knapp, T.E., Mere, L., Burres, N., Feng, L., Whitney, M., Roemer, K. & Tsien, R.Y. (1998) Science 279, 84- 88.

36. Moore, J.T., Davis, S.T. & Dev, I.K. (1997) Anal. Biochem. 247, 203-209.

37. Strynadka, N.C., Jensen, S.E., Johns, K., Blanchard, H., Page, M., Matagne, A., Frere, J.M. & James, M.N. (1994) Nature 368, 657-660.

Sequence Listing

<110>Moyle, William R.

Xing, Yongna

<120> protein nodules

<130>268/279-RWJ-01-40

<140>60/345,283

<141>2001-11-08

<160>56

<170>PatentIn version 3.1

<210>1

<211>92

<212>PRT

<213>Homo sapiens

<400>1

Ala Pro Asp Val Gln Asp Cys Pro Glu Cys Thr Leu Gln Glu Asn Pro

1 5 10 15

Phe Phe Ser Gln Pro Gly Ala Pro Ile Leu Gln Cys Met Gly Cys Cys

20 25 30

Phe Ser Arg Ala Tyr Pro Thr Pro Leu Arg Ser Lys Lys Thr Met Leu

35 40 45

Val Gln Lys Asn Val Thr Ser Glu Ser Thr Cys Cys Val Ala Lys Ser

50 55 60

Tyr Asn Arg Val Thr Val Met Gly Gly Phe Lys Val Glu Asn His Thr

65 70 75 80

Ala Cys His Cys Ser Thr Cys Tyr Tyr His Lys Ser

85 90

<210>2

<211>92

<212>PRT

<213>Artificial Sequence

<220>

<223>hCG alpha-subunit with Cys substituted for Gln5

<400>2

Ala Pro Asp Val Cys Asp Cys Pro Glu Cys Thr Leu Gln Glu Asn Pro

1 5 10 15

Phe Phe Ser Gln Pro Gly Ala Pro Ile Leu Gln Cys Met Gly Cys Cys

20 25 30

Phe Ser Arg Ala Tyr Pro Thr Pro Leu Arg Ser Lys Lys Thr Met Leu

35 40 45

Val Gln Lys Asn Val Thr Ser Glu Ser Thr Cys Cys Val Ala Lys Ser

50 55 60

Tyr Asn Arg Val Thr Val Met Gly Gly Phe Lys Val Glu Asn His Thr

65 70 75 80

Ala Cys His Cys Ser Thr Cys Tyr Tyr His Lys Ser

85 90

<210>3

<211>92

<212>PRT

<213>Artificial Sequence

<220>

<223>hCG alpha-subunit with Cys substituted for Leu12

<400>3

Ala Pro Asp Val Gln Asp Cys Pro Glu Cys Thr Cys Gln Glu Asn Pro

1 5 10 15

Phe Phe Ser Gln Pro Gly Ala Pro Ile Leu Gln Cys Met Gly Cys Cys

20 25 30

Phe Ser Arg Ala Tyr Pro Thr Pro Leu Arg Ser Lys Lys Thr Met Leu

35 40 45

Val Gln Lys Asn Val Thr Ser Glu Ser Thr Cys Cys Val Ala Lys Ser

50 55 60

Tyr Asn Arg Val Thr Val Met Gly Gly Phe Lys Val Glu Asn His Thr

65 70 75 80

Ala Cys His Cys Ser Thr Cys Tyr Tyr His Lys Ser

85 90

<210>4

<211>92

<212>PRT

<213>Artificial Sequence

<220>

<223>hCG alpha-subunit with Cys substituted for Asn15

<400>4

Ala Pro Asp Val Gln Asp Cys Pro Glu Cys Thr Leu Gln Glu Cys Pro

1 5 10 15

Phe Phe Ser Gln Pro Gly Ala Pro Ile Leu Gln Cys Met Gly Cys Cys

20 25 30

Phe Ser Arg Ala Tyr Pro Thr Pro Leu Arg Ser Lys Lys Thr Met Leu

35 40 45

Val Gln Lys Asn Val Thr Ser Glu Ser Thr Cys Cys Val Ala Lys Ser

50 55 60

Tyr Asn Arg Val Thr Val Met Gly Gly Phe Lys Val Glu Asn His Thr

65 70 75 80

Ala Cys His Cys Ser Thr Cys Tyr Tyr His Lys Ser

85 90

<210>5

<211>92

<212>PRT

<213>Artificial Sequence

<220>

<223>hCG alpha-subunit with Cys substituted for Phe17

<400>5

Ala Pro Asp Val Gln Asp Cys Pro Glu Cys Thr Leu Gln Glu Asn Pro

1 5 10 15

Cys Phe Ser Gln Pro Gly Ala Pro Ile Leu Gln Cys Met Gly Cys Cys

20 25 30

Phe Ser Arg Ala Tyr Pro Thr Pro Leu Arg Ser Lys Lys Thr Met Leu

35 40 45

Val Gln Lys Asn Val Thr Ser Glu Ser Thr Cys Cys Val Ala Lys Ser

50 55 60

Tyr Asn Arg Val Thr Val Met Gly Gly Phe Lys Val Glu Asn His Thr

65 70 75 80

Ala Cys His Cys Ser Thr Cys Tyr Tyr His Lys Ser

85 90

<210>6

<211>92

<212>PRT

<213>Artificial Sequence

<220>

<223>hCG alpha-subunit with Cys substituted for Leu22

<400>6

Ala Pro Asp Val Gln Asp Cys Pro Glu Cys Thr Leu Gln Glu Asn Pro

1 5 10 15

Phe Phe Ser Gln Pro Cys Ala Pro Ile Leu Gln Cys Met Gly Cys Cys

20 25 30

Phe Ser Arg Ala Tyr Pro Thr Pro Leu Arg Ser Lys Lys Thr Met Leu

35 40 45

Val Gln Lys Asn Val Thr Ser Glu Ser Thr Cys Cys Val Ala Lys Ser

50 55 60

Tyr Asn Arg Val Thr Val Met Gly Gly Phe Lys Val Glu Asn His Thr

65 70 75 80

Ala Cys His Cys Ser Thr Cys Tyr Tyr His Lys Ser

85 90

<210>7

<211>92

<212>PRT

<213>Artificial Sequence

<220>

<223>hCG alpha-subunit with Cys substituted for Gln7

<400>7

Ala Pro Asp Val Gln Asp Cys Pro Glu Cys Thr Leu Gln Glu Asn Pro

1 5 10 15

Phe Phe Ser Gln Pro Gly Ala Pro Ile Leu Cys Cys Met Gly Cys Cys

20 25 30

Phe Ser Arg Ala Tyr Pro Thr Pro Leu Arg Ser Lys Lys Thr Met Leu

35 40 45

Val Gln Lys Asn Val Thr Ser Glu Ser Thr Cys Cys Val Ala Lys Ser

50 55 60

Tyr Asn Arg Val Thr Val Met Gly Gly Phe Lys Val Glu Asn His Thr

65 70 75 80

Ala Cys His Cys Ser Thr Cys Tyr Tyr His Lys Ser

85 90

<210>8

<211>92

<212>PRT

<213>Artificial Sequence

<220>

<223>hCG alpha-subunit with Cys substituted for Leu22

<400>8

Ala Pro Asp Val Gln Asp Cys Pro Glu Cys Thr Cys Gln Glu Asn Pro

1 5 10 15

Phe Phe Ser Gln Pro Gly Ala Pro Ile Leu Gln Cys Met Gly Cys Cys

20 25 30

Phe Ser Arg Ala Tyr Pro Thr Pro Leu Arg Ser Lys Lys Thr Met Leu

35 40 45

Val Gln Lys Asn Val Thr Ser Glu Ser Thr Cys Cys Val Ala Lys Ser

50 55 60

Tyr Asn Arg Val Thr Val Met Gly Gly Phe Lys Val Glu Asn His Thr

65 70 75 80

Ala Cys His Cys Ser Thr Cys Tyr Tyr His Lys Ser

85 90

<210>9

<211>92

<212>PRT

<213>Artificial Sequence

<220>

<223>hCG alpha-subunit with Cys substituted for Arg35

<400>9

Ala Pro Asp Val Gln Asp Cys Pro Glu Cys Thr Leu Gln Glu Asn Pro

1 5 10 15

Phe Phe Ser Gln Pro Gly Ala Pro Ile Leu Gln Cys Met Gly Cys Cys

20 25 30

Phe Ser Cys Ala Tyr Pro Thr Pro Leu Arg Ser Lys Lys Thr Met Leu

35 40 45

Val Gln Lys Asn Val Thr Ser Glu Ser Thr Cys Cys Val Ala Lys Ser

50 55 60

Tyr Asn Arg Val Thr Val Met Gly Gly Phe Lys Val Glu Asn His Thr

65 70 75 80

Ala Cys His Cys Ser Thr Cys Tyr Tyr His Lys Ser

85 90

<210>10

<211>92

<212>PRT

<213>Artificial Sequence

<220>

<223>hCG alpha-subunit with Cys substituted for Tyr37

<400>10

Ala Pro Asp Va1 Gln Asp Cys Pro Glu Cys Thr Leu Gln Glu Asn Pro

1 5 10 15

Phe Phe Ser Gln Pro Gly Ala Pro Ile Leu Gln Cys Mst Gly Cys Cys

20 25 30

Phe Ser Arg Ala Cys Pro Thr Pro Leu Arg Ser Lys Lys Thr Met Leu

35 40 45

Val Gln Lys Asn Val Thr Ser Glu Ser Thr Cys Cys Val Ala Lys Ser

50 55 60

Tyr Asn Arg Val Thr Val Met Gly Gly Phe Lys Val Glu Asn His Thr

65 70 75 80

Ala Cys His Cys Ser Thr Cys Tyr Tyr His Lys Ser

85 90

<210>11

<211>92

<212>PRT

<213>Artificial Sequence

<220>

<223>hCG alpha-subunit with Cys substituted for Pro38

<400>11

Ala Pro Asp Val Gln Asp Cys Pro Glu Cys Thr Leu Gln Glu Asn Pro

1 5 10 15

Phe Phe Ser Gln Pro Gly Ala Pro Ile Leu Gln Cys Met Gly Cys Cys

20 25 30

Phe Ser Arg Ala Tyr Cys Thr Pro Leu Arg Ser Lys Lys Thr Met Leu

35 40 45

Val Gln Lys Asn Val Thr Ser Glu Ser Thr Cys Cys Val Ala Lys Ser

50 55 60

Tyr Asn Arg Val Thr Val Met Gly Gly Phe Lys Val Glu Asn His Thr

65 70 75 80

Ala Cys His Cys Ser Thr Cys Tyr Tyr His Lys Ser

85 90

<210>12

<211>92

<212>PRT

<213>Artificial Sequence

<220>

<223>hCG alpha-subunit with Cys substituted for Thr39

<400>12

Ala Pro Asp Val Gln Asp Cys Pro Glu Cys Thr Leu Gln Glu Asn Pro

1 5 10 15

Phe Phe Ser Gln Pro Gly Ala Pro Ile Leu Gln Cys Met Gly Cys Cys

20 25 30

Phe Ser Arg Ala Tyr Pro Cys Pro Leu Arg Ser Lys Lys Thr Met Leu

35 40 45

Val Gln Lys Asn Val Thr Ser Glu Ser Thr Cys Cys Val Ala Lys Ser

50 55 60

Tyr Asn Arg Val Thr Val Met Gly Gly Phe Lys Val Glu Asn His Thr

65 70 75 80

Ala Cys His Cys Ser Thr Cys Tyr Tyr His Lys Ser

85 90

<210>13

<211>92

<212>PRT

<213>Artificial Sequence

<220>

<223>hCG alpha-subunit with Cys substituted for Pro40

<400>13

Ala Pro Asp Val Gln Asp Cys Pro Glu Cys Thr Leu Gln Glu Asn Pro

1 5 10 15

Phe Phe Ser Gln Pro Gly Ala Pro Ile Leu Gln Cys Met Gly Cys Cys

20 25 30

Phe Ser Arg Ala Tyr Pro Thr Cys Leu Arg Ser Lys Lys Thr Met Leu

35 40 45

Val Gln Lys Asn Val Thr Ser Glu Ser Thr Cys Cys Val Ala Lys Ser

50 55 60

Tyr Asn Arg Val Thr Val Met Gly Gly Phe Lys Val Glu Asn His Thr

65 70 75 80

Ala Cys His Cys Ser Thr Cys Tyr Tyr His Lys Ser

85 90

<210>14

<211>92

<212>PRT

<213>Artificial Sequence

<220>

<223>hCG alpha-subunit with Cys substituted for Leu41

<400>14

Ala Pro Asp Val Gln Asp Cys Pro Glu Cys Thr Leu Gln Glu Asn Pro

1 5 10 15

Phe Phe Ser Gln Pro Gly Ala Pro Ile Leu Gln Cys Met Gly Cys Cys

20 25 30

Phe Ser Arg Ala Tyr Pro Thr Pro Cys Arg Ser Lys Lys Thr Met Leu

35 40 45

Val Gln Lys Asn Val Thr Ser Glu Ser Thr Cys Cys Val Ala Lys Ser

50 55 60

Tyr Asn Arg Val Thr Val Met Gly Gly Phe Lys Val Glu Asn His Thr

65 70 75 80

Ala Cys His Cys Ser Thr Cys Tyr Tyr His Lys Ser

85 90

<210>15

<211>92

<212>PRT

<213>Artificial Sequence

<220>

<223>hCG alpha-subunit with Cys substituted for Arg42

<400>15

Ala Pro Asp Val Gln Asp Cys Pro Glu Cys Thr Leu Gln Glu Asn Pro

1 5 10 15

Phe Phe Ser Gln Pro Gly Ala Pro Ile Leu Gln Cys Met Gly Cys Cys

20 25 30

Phe Ser Arg Ala Tyr Pro Thr Pro Leu Cys Ser Lys Lys Thr Met Leu

35 40 45

Val Gln Lys Asn Val Thr Ser Glu Ser Thr Cys Cys Val Ala Lys Ser

50 55 60

Tyr Asn Arg Val Thr Val Met Gly Gly Phe Lys Val Glu Asn His Thr

65 70 75 80

Ala Cys His Cys Ser Thr Cys Tyr Tyr His Lys Ser

85 90

<210>16

<211>92

<212>PRT

<213>Artificial Sequence

<220>

<223>hCG alpha-subunit with Cys substituted for Ser43

<400>16

Ala Pro Asp Val Gln Asp Cys Pro Glu Cye Thr Leu Gln Glu Asn Pro

1 5 10 15

Phe Phe Ser Gln Pro Gly Ala Pro Ile Leu Gln Cys Met Gly Cys Cys

20 25 30

Phe Ser Arg Ala Tyr Pro Thr Pro Leu Arg Cys Lys Lys Thr Met Leu

35 40 45

Val Gln Lys Asn Val Thr Ser Glu Ser Thr Cys Cys Val Ala Lys Ser

50 55 60

Tyr Asn Arg Val Thr Val Met Gly Gly Phe Lys Val Glu Asn His Thr

65 70 75 80

Ala Cys His Cys Ser Thr Cys Tyr Tyr His Lys Ser

85 90

<210>17

<211>92

<212>PRT

<213>Artificial Sequence

<220>

<223>hCG alpha-subunit with Cys substituted for Lys44

<400>17

Ala Pro Asp Val Gln Asp Cys Pro Glu Cys Thr Leu Gln Glu Asn Pro

1 5 10 15

Phe Phe Ser Gln Pro Gly Ala Pro Ile Leu Gln Cys Met Gly Cys Cys

20 25 30

Phe Ser Arg Ala Tyr Pro Thr Pro Leu Arg Ser Cys Lys Thr Met Leu

35 40 45

Val Gln Lys Asn Val Thr Ser Glu Ser Thr Cys Cys Val Ala Lys Ser

50 55 60

Tyr Asn Arg Val Thr Val Met Gly Gly Phe Lys Val Glu Asn His Thr

65 70 75 80

Ala Cys His Cys Ser Thr Cys Tyr Tyr His Lys Ser

85 90

<210>18

<211>92

<212>PRT

<213>Artificial Sequence

<220>

<223>hCG alpha-subunit with Cys substituted for Lys45

<400>18

Ala Pro Asp Val Gln Asp Cys Pro Glu Cys Thr Leu Gln Glu Asn Pro

1 5 10 15

Phe Phe Ser Gln Pro Gly Ala Pro Ile Leu Gln Cys Met Gly Cys Cys

20 25 30

Phe Ser Arg Ala Tyr Pro Thr Pro Leu Arg Ser Lys Cys Thr Met Leu

35 40 45

Val Gln Lys Asn Val Thr Ser Glu Ser Thr Cys Cys Val Ala Lys Ser

50 55 60

Tyr Asn Arg Val Thr Val Met Gly Gly Phe Lys Val Glu Asn His Thr

65 70 75 80

Ala Cys His Cys Ser Thr Cys Tyr Tyr His Lys Ser

85 90

<210>19

<211>92

<212>PRT

<213>Artificial Sequence

<220>

<223>hCG alpha subunit with Cys substituted for Thr46

<400>19

Ala Pro Asp Val Gln Asp Cys Pro Glu Cys Thr Leu Gln Glu Asn Pro

1 5 10 15

Phe Phe Ser Gln Pro Gly Ala Pro Ile Leu Gln Cys Met Gly Cys Cys

20 25 30

Phe Ser Arg Ala Tyr Pro Thr Pro Leu Arg Ser Lys Lys Cys Met Leu

35 40 45

Val Gln Lys Asn Val Thr Ser Glu Ser Thr Cys Cys Val Ala Lys Ser

50 55 60

Tyr Asn Arg Val Thr Val Met Gly Gly Phe Lys Val Glu Asn His Thr

65 70 75 80

Ala Cys His Cys Ser Thr Cys Tyr Tyr His Lys Ser

85 90

<210>20

<211>92

<212>PRT

<213>Artificial Sequence

<220>

<223>hCG alpha-subunit with Cys substituted for Met47

<400>20

Ala Pro Asp Val Gln Asp Cys Pro Glu Cys Thr Leu Gln Glu Asn Pro

1 5 10 15

Phe Phe Ser Gln Pro Gly Ala Pro Ile Leu Gln Cys Met Gly Cys Cys

20 25 30

Phe Ser Arg Ala Tyr Pro Thr Pro Leu Arg Ser Lys Lys Thr Cys Leu

35 40 45

Val Gln Lys Asn Val Thr Ser Glu Ser Thr Cys Cys Val Ala Lys Ser

50 55 60

Tyr Asn Arg Val Thr Val Met Gly Gly Phe Lys Val Glu Asn His Thr

65 70 75 80

Ala Cys His Cys Ser Thr Cys Tyr Tyr His Lys Ser

85 90

<210>21

<211>92

<212>PRT

<213>Artificial Sequence

<220>

<223>hCG alpha-subunit with Cys substituted for Leu48

<400>21

Ala Pro Asp Val Gln Asp Cys Pro Glu Cys Thr Leu Gln Glu Asn Pro

1 5 10 15

Phe Phe Ser Gln Pro Gly Pra Pro Ile Leu Gln Cys Met Gly Cys Cys

20 25 30

Phe Ser Arg Ala Tyr Pro Thr Pro Leu Arg Ser Lys Lys Thr Met Cys

35 40 45

Val Gln Lys Asn Val Thr Ser Glu Ser Thr Cys Cys Val Ala Lys Ser

50 55 60

Tyr Asn Arg Val Thr Val Met Gly Gly Phe Lys Val Glu Asn His Thr

65 70 75 80

Ala Cys His Cys Ser Thr Cys Tyr Tyr His Lys Ser

85 90

<210>22

<211>92

<212>PRT

<213>Artificial Sequence

<220>

<223>hCG alpha-subunit with Cys substituted for Val49

<400>22

Ala Pro Asp Val Gln Asp Cys Pro Glu Cys Thr Leu Gln Glu Asn Pro

1 5 10 15

Phe Phe Ser Gln Pro Gly Ala Pro Ile Leu Gln Cys Met Gly Cys Cys

20 25 30

Phe Ser Arg Ala Tyr Pro Thr Pro Leu Arg Ser Lys Lys Thr Met Leu

35 40 45

Cys Gln Lys Asn Val Thr Ser Glu Ser Thr Cys Cys Val Ala Lys Ser

50 55 60

Tyr Asn Arg Val Thr Val Met Gly Gly Phe Lys Val Glu Asn His Thr

65 70 75 80

Ala Cys His Cys Ser Thr Cys Tyr Tyr His Lys Ser

85 90

<210>23

<211>92

<212>PRT

<213>Artificial Sequence

<220>

<223>hCG alpha-subunit with Cys substituted for Gln50

<400>23

Ala Pro Asp Val Gln Asp Cys Pro Glu Cys Thr Leu Gln Glu Asn Pro

1 5 10 15

Phe Phe Ser Gln Pro Gly Ala Pro Ile Leu Gln Cys Met Gly Cys Cys

20 25 30

Phe Ser Arg Ala Tyr Pro Thr Pro Leu Arg Ser Lys Lys Thr Met Leu

35 40 45

Val Cys Lys Asn Val Thr Ser Glu Ser Thr Cys Cys Val Ala Lys Ser

50 55 60

Tyr Asn Arg Val Thr Val Met Gly Gly Phe Lys Val Glu Asn His Thr

65 70 75 80

Ala Cys His Cys Ser Thr Cys Tyr Tyr His Lys Ser

85 90

<210>24

<211>92

<212>PRT

<213>Artificial Sequence

<220>

<223>hCG alpha-subunit with Cys substituted for Lys51

<400>24

Ala Pro Asp Val Gln Asp Cys Pro Glu Cys Thr Leu Gln Glu Asn Pro

1 5 10 15

Phe Phe Ser Gln Pro Gly Ala Pro Ile Leu Gln Cys Met Gly Cys Cys

20 25 30

Phe Ser Arg Ala Tyr Pro Thr Pro Leu Arg Ser Lys Lys Thr Met Leu

35 40 45

Val Gln Cys Asn Val Thr Ser Glu Ser Thr Cys Cys Val Ala Lys Ser

50 55 60

Tyr Asn Arg Val Thr Val Met Gly Gly Phe Lys Val Glu Asn His Thr

65 70 75 80

Ala Cys His Cys Ser Thr Cys Tyr Tyr His Lys Ser

85 90

<210>25

<211>92

<212>PRT

<213>Artificial Sequence

<220>

<223>hCG alpha-subunit with Cys substituted for Asn52

<400>25

Ala Pro Asp Val Gln Asp Cys Pro Glu Cys Thr Leu Gln Glu Asn Pro

1 5 10 15

Phe Phe Ser Gln Pro Gly Ala Pro Ile Leu Gln Cys Met Gly Cys Cys

20 25 30

Phe Ser Arg Ala Tyr Pro Thr Pro Leu Arg Ser Lys Lys Thr Met Leu

35 40 45

Val Gln Lys Cys Val Thr Ser Glu Ser Thr Cys Cys Val Ala Lys Ser

50 55 60

Tyr Asn Arg Val Thr Val Met Gly Gly Phe Lys Val Glu Asn His Thr

65 70 75 80

Ala Cys His Cys Ser Thr Cys Tyr Tyr His Lys Ser

85 90

<210>26

<211>92

<212>PRT

<213>Artificial Sequence

<220>

<223>hCG alpha-subunit with Cys substituted for Val53

<400>26

Ala Pro Asp Val Gln Asp Cys Pro Glu Cys Thr Leu Gln Glu Asn Pro

1 5 10 15

Phe Phe Ser Gln Pro Gly Ala Pro Ile Leu Gln Cys Met Gly Cys Cys

20 25 30

Phe Ser Arg Ala Tyr Pro Thr Pro Leu Arg Ser Lys Lys Thr Met Leu

35 40 45

Val Gln Lys Asn Cys Thr Ser Glu Ser Thr Cys Cys Val Ala Lys Ser

50 55 60

Tyr Asn Arg Val Thr Val Met Gly Gly Phe Lys Val Glu Asn His Thr

65 70 75 80

Ala Cys His Cys Ser Thr Cys Tyr Tyr His Lys Ser

85 90

<210>27

<211>92

<212>PRT

<213>Artificial Sequence

<220>

<223>hCG alpha-subunit with Cys substituted for Glu56

<400>27

Ala Pro Asp Val Gln Asp Cys Pro Glu Cys Thr Leu Gln Glu Asn Pro

1 5 10 15

Phe Phe Ser Gln Pro Gly Ala Pro Ile Leu Gln Cys Met Gly Cys Cys

20 25 30

Phe Ser Arg Ala Tyr Pro Thr Pro Leu Arg Ser Lys Lys Thr Met Leu

35 40 45

Val Gln Lys Asn Val Thr Ser Cys Ser Thr Cys Cys Val Ala Lys Ser

50 55 60

Tyr Asn Arg Val Thr Val Met Gly Gly Phe Lys Val Glu Asn His Thr

65 70 75 80

Ala Cys His Cys Ser Thr Cys Tyr Tyr His Lys Ser

85 90

<210>28

<211>92

<212>PRT

<213>Artificial Sequence

<220>

<223>hCG alpha-subunit with Cys substituted for Ser64

<400>28

Ala Pro Asp Val Gln Asp Cys Pro Glu Cys Thr Leu Gln Glu Asn Pro

1 5 10 15

Phe Phe Ser Gln Pro Gly Ala Pro Ile Leu Gln Cys Met Gly Cys Cys

20 25 30

Phe Ser Arg Ala Tyr Pro Thr Pro Leu Arg Ser Lys Lys Thr Met Leu

35 40 45

Val Gln Lys Asn Val Thr Ser Glu Ser Thr Cys Cys Val Ala Cys Ser

50 55 60

Tyr Asn Arg Val Thr Val Met Gly Gly Phe Lys Val Glu Asn His Thr

65 70 75 80

Ala Cys His Cys Ser Thr Cys Tyr Tyr His Lys Ser

85 90

<210>29

<211>92

<212>PRT

<213>Artificial Sequence

<220>

<223>hCG alpha-subunit with Cys substituted for Val76

<400>29

Ala Pro Asp Val Gln Asp Cys Pro Glu Cys Thr Leu Gln Glu Asn Pro

1 5 10 15

Phe Phe Ser Gln Pro Gly Ala Pro Ile Leu Gln Cys Met Gly Cys Cys

20 25 30

Phe Ser Arg Ala Tyr Pro Thr Pro Leu Arg Ser Lys Lys Thr Met Leu

35 40 45

Val Gln Lys Asn Val Thr Ser Glu Ser Thr Cys Cys Val Ala Lys Ser

50 55 60

Tyr Asn Arg Val Thr Val Met Gly Gly Phe Lys Cys Glu Asn His Thr

65 70 75 80

Ala Cys His Cys Ser Thr Cys Tyr Tyr His Lys Ser

85 90

<210>30

<211>92

<212>PRT

<213>Artificial Sequence

<220>

<223>hCG alpha-subunit with Cys substituted for Thr86

<400>30

Ala Pro Asp Val Gln Asp Cys Pro Glu Cys Thr Leu Gln Glu Asn Pro

1 5 10 15

Phe Phe Ser Gln Pro Gly Ala Pro Ile Leu Gln Cys Met Gly Cys Cys

20 25 30

Phe Ser Arg Ala Tyr Pro Thr Pro Leu Arg Ser Lys Lys Thr Met Leu

35 40 45

Val Gln Lys Asn Val Thr Ser Glu Ser Thr Cys Cys Val Ala Lys Ser

50 55 60

Tyr Asn Arg Val Thr Val Met Gly Gly Phe Lys Val Glu Asn His Thr

65 70 75 80

Ala Cys His Cys Ser Cys Cys Tyr Tyr His Lys Ser

85 90

<210>31

<211>92

<212>PRT

<213>Artificial Sequence

<220>

<223>hCG alpha-subunit with Cys substituted for Tyr88

<400>31

Ala Pro Asp Val Gln Asp Cys Pro Glu Cys Thr Leu Gln Glu Asn Pro

1 5 10 15

Phe Phe Ser Gln Pro Gly Ala Pro Ile Leu Gln Cys Met Gly Cys Cys

20 25 30

Phe Ser Arg Ala Tyr Pro Thr Pro Leu Arg Ser Lys Lys Thr Met Leu

35 40 45

Val Gln Lys Asn Val Thr Ser Glu Ser Thr Cys Cys Val Ala Lys Ser

50 55 60

Tyr Asn Arg Val Thr Val Met Gly Gly Phe Lys Val Glu Asn His Thr

65 70 75 80

Ala Cys His Cys Ser Thr Cys Cys Tyr His Lys Ser

85 90

<210>32

<211>92

<212>PRT

<213>Artificial Sequence

<220>

<223>hCG alpha-subunit with Cys substituted for Leu89

<400>32

Ala Pro Asp Val Gln Asp Cys Pro Glu Cys Thr Leu Gln Glu Asn Pro

1 5 10 15

Phe Phe Ser Gln Pro Gly Ala Pro Ile Leu Gln Cys Met Gly Cys Cys

20 25 30

Phe Ser Arg Ala Tyr Pro Thr Pro Leu Arg Ser Lys Lys Thr Met Leu

35 40 45

Val Gln Lys Asn Val Thr Ser Glu Ser Thr Cys Cys Val Ala Lys Ser

50 55 60

Tyr Asn Arg Val Thr Val Met Gly Gly Phe Lys Val Glu Asn His Thr

65 70 75 80

Ala Cys His Cys Ser Thr Cys Tyr Cys His Lys Ser

85 90

<210>33

<211>92

<212>PRT

<213>Artificial Sequence

<220>

<223>hCG alpha-subunit with Cys substituted for His90

<400>33

Ala Pro Asp Val Gln Asp Cys Pro Glu Cys Thr Leu Gln Glu Asn Pro

1 5 10 15

Phe Phe Ser Gln Pro Gly Ala Pro Ile Leu Gln Cys Met Gly Cys Cys

20 25 30

Phe Ser Arg Ala Tyr Pro Thr Pro Leu Arg Ser Lys Lys Thr Met Leu

35 40 45

Val Gln Lye Asn Val Thr Ser Glu Ser Thr Cys Cys Val Ala Lys Ser

50 55 60

Tyr Asn Arg Val Thr Val Met Gly Gly Phe Lys Val Glu Asn His Thr

65 70 75 80

Ala Cys His Cys Ser Thr Cys Tyr Tyr Cys Lys Ser

85 90

<210>34

<211>92

<212>PRT

<213>Artificial Sequence

<220>

<223>hCG alpha-subunit with Cys substituted for Lys91

<400>34

Ala Pro Asp Val Gln Asp Cys Pro Glu Cys Thr Leu Gln Glu Asn Pro

1 5 10 15

Phe Phe Ser Gln Pro Gly Ala Pro Ile Leu Gln Cys Met Gly Cys Cys

20 25 30

Phe Ser Arg Ala Tyr Pro Thr Pro Leu Arg Ser Lys Lys Thr Met Leu

35 40 45

Val Gln Lys Asn Val Thr Ser Glu Ser Thr Cys Cys Val Ala Lys Ser

50 55 60

Tyr Asn Arg Val Thr Val Met Gly Gly Phe Lys Val Glu Asn His Thr

65 70 75 80

Ala Cys His Cys Ser Thr Cys Tyr Tyr His Cys Ser

85 90

<210>35

<211>92

<212>PRT

<213>Artificial Sequence

<220>

<223>hCG alpha-subunit with Cys substituted for Ser92

<400>35

Ala Pro Asp Val Gln Asp Cys Pro Glu Cys Thr Leu Gln Glu Asn Pro

1 5 10 15

Phe Phe Ser Gln Pro Gly Ala Pro Ile Leu Gln Cys Met Gly Cys Cys

20 25 30

Phe Ser Arg Ala Tyr Pro Thr Pro Leu Arg Ser Lys Lys Thr Met Leu

35 40 45

Val Gln Lys Asn Val Thr Ser Glu Ser Thr Cys Cys Val Ala Lys Ser

50 55 60

Tyr Asn Arg Val Thr Val Met Gly Gly Phe Lys Val Glu Asn His Thr

65 70 75 80

Ala Cys His Cys Ser Thr Cys Tyr Tyr His Lys Cys

85 90

<210>36

<211>145

<212>PRT

<213>Homo sapiens

<400>36

Ser Lys Glu Pro Leu Arg Pro Arg Cys Arg Pro Ile Asn Ala Thr Leu

1 5 10 15

Ala Val Glu Lys Glu Gly Cys Pro Val Cys Ile Thr Val Asn Thr Thr

20 25 30

Ile Cys Ala Gly Tyr Cys Pro Thr Met Thr Arg Val Leu Gln Gly Val

35 40 45

Leu Pro Ala Leu Pro Gln Val Val Cys Asn Tyr Arg Asp Val Arg Phe

50 55 60

Glu Ser Ile Arg Leu Pro Gly Cys Pro Arg Gly Val Asn Pro Val Val

65 70 75 80

Ser Tyr Ala Val Ala Leu Ser Cys Gln Cys Ala Leu Cys Arg Arg Ser

85 90 95

Thr Thr Asp Cys Gly Gly Pro Lys Asp His Pro Leu Thr Cys Asp Asp

100 105 110

Pro Arg Phe Gln Asp Ser Ser Ser Ser Ser Lys Ala Pro Pro Pro Ser Leu

115 120 125

Pro Ser Pro Ser Arg Leu Pro Gly Pro Ser Asp Thr Pro Ile Leu Pro Gln

130 135 140

<210>37

<211>145

<212>PRT

<213>Artificial Sequence

<220>

<223>hCG beta-subunit with Cys substituted for Ser138

<400>37

Ser Lys Glu Pro Leu Arg Pro Arg Cys Arg Pro Ile Asn Ala Thr Leu

1 5 10 15

Ala Val Glu Lys Glu Gly Cys Pro Val Cys Ile Thr Val Asn Thr Thr

20 25 30

Ile Cys Ala Gly Tyr Cys Pro Thr Met Thr Arg Val Leu Gln Gly Val

35 40 45

Leu Pro Ala Leu Pro Gln Val Val Cys Asn Tyr Arg Asp Val Arg Phe

50 55 60

Glu Ser Ile Arg Leu Pro Gly Cys Pro Arg Gly Val Asn Pro Val Val

65 70 75 80

Ser Tyr Ala Val Ala Leu Ser Cys Gln Cys Ala Leu Cys Arg Arg Ser

85 90 95

Thr Thr Asp Cys Gly Gly Pro Lys Asp His Pro Leu Thr Cys Asp Asp

100 105 110

Pro Arg Phe Gln Asp Ser Ser Ser Ser Ser Lys Ala Pro Pro Pro Ser Leu

115 120 125

Pro Ser Pro Ser Arg Leu Pro Gly Pro Cys Asp Thr Pro Ile Leu Pro Gln

130 135 140

<210>38

<211>145

<212>PRT

<213>Artificial Sequence

<220>

<223>hCG beta-subunit residues 101-114 were replaced with their hFSH b

eta-subunit counterparts, namely hFSH beta-subunit residues 95-10

8

<400>38

Ser Lys Glu Pro Leu Arg Pro Arg Cys Arg Pro Ile Asn Ala Thr Leu

1 5 10 15

Ala Val Glu Lys Glu Gly Cys Pro Val Cys Ile Thr Val Asn Thr Thr

20 25 30

Ile Cys Ala Gly Tyr Cys Pro Thr Met Thr Arg Val Leu Gln Gly Val

35 40 45

Leu Pro Ala Leu Pro Gln Val Val Cys Asn Tyr Arg Asp Val Arg Phe

50 55 60

Glu Ser Ile Arg Leu Pro Gly Cys Pro Arg Gly Val Pro Asn Val Val

65 70 75 80

Ser Tyr Ala Val Ala Leu Ser Cys Gln Cys Ala Leu Cys Arg Arg Ser

85 90 95

Thr Thr Asp Cys Thr Val Arg Gly Leu Gly Pro Ser Tyr Cys Ser Phe

100 105 110

Gly Glu Phe Gln Asp Ser Ser Ser Ser Ser Lys Ala Pro Pro Pro Ser Leu

115 120 125

Pro Ser Pro Ser Arg Leu Pro Gly Pro Ser Asp Thr Pro Ile Leu Pro Gln

130 135 140

<210>39

<211>145

<212>PRT

<213>Artificial Sequence

<220>

<223>hCG beta-subunit residues 101-114 were replaced with their hFSH b

eta-subunit counterparts, namely hFSH beta-subunit residues 95-10

8, and Serine38 in the beta-subunit carboxyterminus of this

Analog was replaced with Cys

<400>39

Ser Lys Glu Pro Leu Arg Pro Arg Cys Arg Pro Ile Asn Ala Thr Leu

1 5 10 15

Ala Val Glu Lys Glu Gly Cys Pro Val Cys Ile Thr Val Asn Thr Thr

20 25 30

Ile Cys Ala Gly Tyr Cys Pro Thr Met Thr Arg Val Leu Gln Gly Val

35 40 45

Leu Pro Ala Leu Pro Gln Val Val Cys Asn Tyr Arg Asp Val Arg Phe

50 55 60

Glu Ser Ile Arg Leu Pro Gly Cys Pro Arg Gly Val Pro Asn Val Val

65 70 75 80

Ser Tyr Ala Val Ala Leu Ser Cys Gln Cys Ala Leu Cys Arg Arg Ser

85 90 95

Thr Thr Asp Cys Thr Val Arg Gly Leu Gly Pro Ser Tyr Cys Ser Phe

100 105 110

Gly Glu Phe Gln Asp Ser Ser Ser Ser Ser Lys Ala Pro Pro Pro Ser Leu

115 120 125

Pro Ser Pro Ser Arg Leu Pro Gly Pro Cys Asp Thr Pro Ile Leu Pro Gln

130 135 140

<210>40

<211>111

<212>PRT

<213>Homos sapiens

<400>40

Asn Ser Cys Glu Leu Thr Asn Ile Thr Ile Ala Val Glu Lys Glu Gly

1 5 10 15

Cys Gly Phe Cys Ile Thr Ile Asn Thr Thr Trp Cys Ala Gly Tyr Cys

20 25 30

Tyr Thr Arg Asp Leu Val Tyr Lys Asp Pro Ala Arg Pro Lys Ile Gln

35 40 45

Lys Thr Cys Thr Phe Lys Glu Leu Val Tyr Glu Thr Val Arg Val Pro

50 55 60

Gly Cys Ala His His Ala Asp Ser Leu Tyr Thr Tyr Pro Val Ala Thr

65 70 75 80

Gln Cys His Cys Gly Lys Cys Asp Ser Asp Ser Thr Asp Cys Thr Val

85 90 95

Arg Gly Leu Gly Pro Ser Tyr Cys Ser Phe Gly Glu Met Lys Glu

100 105 110

<210>41

<211>139

<212>PRT

<213>Artificial Sequence

<220>

<223>hFSH beta-subunit analog lacking the leader peptide of hFSH beta-

subunit with hFSH residues 1-108 and hCG residues 115-145 in

tandem

<400>41

Asn Ser Cys Glu Leu Thr Asn Ile Thr Ile Ala Val Glu Lys Glu Gly

1 5 10 15

Cys Gly Phe Cys Ile Thr Ile Asn Thr Thr Trp Cys Ala Gly Tyr Cys

20 25 30

Tyr Thr Arg Asp Leu Val Tyr Lys Asp Pro Ala Arg Pro Lys Ile Gln

35 40 45

Lys Thr Cys Thr Phe Lys Glu Leu Val Tyr Glu Thr Val Arg Val Pro

50 55 60

Gly Cys Ala His His Ala Asp Ser Leu Tyr Thr Tyr Pro Val Ala Thr

65 70 75 80

Gln Cys His Cys Gly Lys Cys Asp Ser Asp Ser Thr Asp Cys Thr Val

85 90 95

Arg Gly Leu Gly Pro Ser Tyr Cys Ser Phe Gly Glu Phe Gln Asp Ser

100 105 110

Ser Ser Ser Lys Ala Pro Pro Pro Ser Leu Pro Ser Pro Ser Arg Leu

115 120 125

Pro Gly Pro Ser Asp Thr Pro Ile Leu Pro Gln

130 135

<210>42

<211>137

<212>PRT

<213>Artificial Sequence

<220>

<223>hFSH beta-subunit analog lacking the leader peptide of hFSH beta-

subunit with hFSH residues 1-108 and hCG residues 115-145 in tandem

em and with Ser132 replaced with Cys

<400>42

Asn Ser Cys Glu Leu Thr Asn Ile Thr Ile Ala Val Glu Lys Glu Gly

1 5 10 15

Cys Gly Phe Cys Ile Thr Ile Asn Thr Thr Trp Cys Ala Gly Tyr Cys

20 25 30

Tyr Thr Arg Asp Leu Val Tyr Lys Asp Pro Ala Arg Pro Lys Ile Gln

35 40 45

Lys Thr Cys Thr Phe Lys Glu Leu Val Tyr Glu Thr Val Arg Val Pro

50 55 60

Gly Cys Ala His His Ala Asp Ser Leu Tyr Thr Tyr Pro Val Ala Thr

65 70 75 80

Gln Cys His Cys Gly Lys Cys Asp Ser Asp Ser Thr Asp Cys Thr Val

85 90 95

Arg Gly Leu Gly Pro Ser Tyr Cys Ser Phe Gly Glu Phe Gln Asp Ser

100 105 110

Ser Ser Ser Lys Ala Pro Pro Pro Ser Leu Pro Ser Pro Ser Arg Leu

115 120 125

Pro Gly Pro Cys Asp Thr Pro Ile Leu

130 135

<210>43

<211>401

<212>PRT

<213>Artificial Sequence

<220>

<223>hCGbeta, S138C-betaLA(short), beta-lactamase fused to a truncated

Version of hCGbeta, S138C

<400>43

Ser Lys Glu Pro Leu Arg Pro Arg Cys Arg Pro Ile Asn Ala Thr Leu

1 5 10 15

Ala Val Glu Lys Glu Gly Cys Pro Val Cys Ile Thr Val Asn Thr Thr

20 25 30

Ile Cys Ala Gly Tyr Cys Pro Thr Met Thr Arg Val Leu Gln Gly Val

35 40 45

Leu Pro Ala Leu Pro Gln Val Val Cys Asn Tyr Arg Asp Val Arg Phe

50 55 60

Glu Ser Ile Arg Leu Pro Gly Cys Pro Arg Gly Val Asn Pro Val Val

65 70 75 80

ser Tyr Ala Val Ala Leu Ser Cys Gln Cys Ala Leu Cys Arg Arg Ser

85 90 95

Thr Thr Asp Cys Gly Gly Pro Lys Asp His Pro Leu Thr Cys Asp Asp

100 105 110

Pro Arg Phe Gln Asp Ser Ser Ser Ser Ser Lys Ala Pro Pro Pro Ser Leu

115 120 125

Pro Ser Pro Ser Arg Leu Pro Gly Pro Cys Asp His Pro Glu Thr Leu

130 135 140

Val Lys Val Lys Asp Ala Glu Asp Gln Leu Gly Ala Arg Val Gly Tyr

145 150 155 160

Ile Glu Leu Asp Leu Asn Ser Gly Lys Ile Leu Glu Ser Phe Arg Pro

165 170 175

Glu Glu Arg Phe Pro Met Met Ser Thr Phe Lys Val Leu Leu Cys Gly

180 185 190

Ala Val Leu Ser Arg Ile Asp Ala Gly Gln Glu Gln Leu Gly Arg Arg

195 200 205

Ile His Tyr Ser Gln Asn Asp Leu Val Glu Tyr Ser Pro Val Thr Glu

210 215 220

Lys His Leu Thr Asp Gly Met Thr Val Arg Glu Leu Cys Ser Ala Ala

225 230 235 240

Ile Thr Met Ser Asp Asn Thr Ala Ala Asn Leu Leu Leu Thr Thr Ile

245 250 255

Gly Gly Pro Lys Glu Leu Thr Ala Phe Leu His Asn Met Gly Asp His

260 265 270

Val Thr Arg Leu Asp Arg Trp Glu Pro Glu Leu Asn Glu Ala Ile Pro

275 280 285

Asn Glu Arg Asp Thr Thr Met Pro Val Ala Met Ala Thr Thr Leu Arg

290 295 300

Lys Leu Leu Thr Gly Glu Leu Leu Thr Leu Ala Ser Arg Gln Gln Leu

305 310 315 320

Ile Asp Trp Met Glu Ala Asp Lys Val Ala Gly Pro Leu Leu Arg Ser

325 330 335

Ala Leu Pro Ala Gly Trp Phe Ile Ala Asp Lys Ser Gly Ala Gly Glu

340 345 350

Arg Gly Ser Arg Gly Ile Ile Ala Ala Leu Gly Pro Asp Gly Lys Pro

355 360 365

Ser Arg Ile Val Val Ile Tyr Thr Thr Gly Ser Gln Ala Thr Met Asp

370 375 380

Glu Arg Asn Arg Gln Ile Ala Glu Ile Gly Ala Ser Leu Ile Lys His

385 390 395 400

Trp

<210>44

<211>408

<212>PRT

<213>Artificial Sequence

<220>

<223>hCGbeta, S138C-betaLA(long), beta-lactamase fused to the carboxyte

rminal end of hCGb, S138C

<400>44

Ser Lys Glu Pro Leu Arg Pro Arg Cys Arg Pro Ile Asn Ala Thr Leu

1 5 10 15

Ala Val Glu Lys Glu Gly Cys Pro Val Cys Ile Thr Val Asn Thr Thr

20 25 30

Ile Cys Ala Gly Tyr Cys Pro Thr Met Thr Arg Val Leu Gln Gly Val

35 40 45

Leu Pro Ala Leu Pro Gln Val Val Cys Asn Tyr Arg Asp Val Arg Phe

50 55 60

Glu Ser Ile Arg Leu Pro Gly Cys Pro Arg Gly Val Asn Pro Val Val

65 70 75 80

Ser Tyr Ala Val Ala Leu Ser Cys Gln Cys Ala Leu Cys Arg Arg Ser

85 90 95

Thr Thr Asp Cys Gly Gly Pro Lys Asp His Pro Leu Thr Cys Asp Asp

100 105 110

Pro Arg Phe Gln Asp Ser Ser Ser Ser Ser Lys Ala Pro Pro Pro Ser Leu

115 120 125

Pro Ser Pro Ser Arg Leu Pro Gly Pro Cys Asp Thr Pro Ile Leu Pro

130 135 140

Gln His Pro Glu Thr Leu Val Lys Val Lys Asp Ala Glu Asp Gln Leu

145 150 155 160

Gly Ala Arg Val Gly Tyr Ile Glu Leu Asp Leu Asn Ser Gly Lys Ile

165 170 175

Leu Glu Ser Phe Arg Pro Glu Glu Arg Phe Pro Met Met Ser Thr Phe

180 185 190

Lys Val Leu Leu Cys Gly Ala Val Leu Ser Arg Ile Asp Ala Gly Gln

195 200 205

Glu Gln Leu Gly Arg Arg Ile His Tyr Ser Gln Asn Asp Leu Val Glu

210 215 220

Tyr Ser Pro Val Thr Glu Lys His Leu Thr Asp Gly Met Thr Val Arg

225 230 235 240

Glu Leu Cys Ser Ala Ala Ile Thr Met Ser Asp Asn Thr Ala Ala Asn

245 250 255

Leu Leu Leu Thr Thr Ile Gly Gly Pro Lys Glu Leu Thr Ala Phe Leu

260 265 270

His Asn Met Gly Asp His Val Thr Arg Leu Asp Arg Trp Glu Pro Glu

275 280 285

Leu Asn Glu Ala Ile Pro Asn Asp Glu Arg Asp Thr Thr Met Pro Val

290 295 300

Ala Met Ala Thr Thr Leu Arg Lys Leu Leu Thr Gly Glu Leu Leu Thr

305 310 315 320

Leu Ala Ser Arg Gln Gln Leu Ile Asp Trp Met Glu Ala Asp Lys Val

325 330 335

Ala Gly Pro Leu Leu Arg Ser Ala Leu Pro Ala Gly Trp Phe Ile Ala

340 345 350

Asp Lys Ser Gly Ala Gly Glu Arg Gly Ser Arg Gly Ile Ile Ala Ala

355 360 365

Lsu Gly Pro Asp Gly Lys Pro Ser Arg Ile Val Val Ile Tyr Thr Thr

370 375 380

Gly Ser Gln Ala Thr Met Asp Glu Arg Aen Arg Gln Ile Ala Glu Ile

385 390 395 400

Gly Ala Ser Leu Ile Lys His Trp

405

<210>45

<211>125

<212>PRT

<213>Artificial Sequence

<220>

<223> hCGbeta, delta116-135, S138C

<400>45

Ser Lys Glu Pro Leu Arg Pro Arg Cys Arg Pro Ile Asn Ala Thr Leu

1 5 10 15

Ala Val Glu Lys Glu Gly Cys Pro Val Cys Ile Thr Val Asn Thr Thr

20 25 30

Ile Cys Ala Gly Tyr Cys Pro Thr Met Thr Arg Val Leu Gln Gly Val

35 40 45

Leu Pro Ala Leu Pro Gln Val Val Cys Asn Tyr Arg Asp Val Arg Phe

50 55 60

Glu Ser Ile Arg Leu Pro Gly Cys Pro Arg Gly Val Asn Pro Val Val

65 70 75 80

Ser Tyr Ala Val Ala Leu Ser Cys Gln Cys Ala Leu Cys Arg Arg Ser

85 90 95

Thr Thr Asp Cys Gly Gly Pro Lys Asp His Pro Leu Thr Cys Asp Asp

100 105 110

Pro Arg Phe Gly Pro Cys Asp Thr Pro Ile Leu Pro Gln

115 120

<210>46

<211>130

<212>PRT

<213>Artificial Sequence

<220>

<223> hCGbeta, delta121-135, S138C

<400>46

Ser Lys Glu Pro Leu Arg Pro Arg Cys Arg Pro Ile Asn Ala Thr Leu

1 5 10 15

Ala Val Glu Lys Glu Gly Cys Pro Val Cys Ile Thr Val Asn Thr Thr

20 25 30

Ile Cys Ala Gly Tyr Cys Pro Thr Met Thr Arg Val Leu Gln Gly Val

35 40 45

Leu Pro Ala Leu Pro Gln Val Val Cys Asn Tyr Ars Asp Val Arg Phe

50 55 60

Glu Ser Ile Arg Leu Pro Gly Cys Pro Arg Gly Val Asn Pro Val Val

65 70 75 80

Ser Tyr Ala Val Ala Leu Ser Cys Gln Cys Ala Leu Cys Arg Arg Ser

85 90 95

Thr Thr Aep Cys Gly Gly Pro Lys Asp His Pro Leu Thr Cys Asp Asp

100 105 110

Pro Arg Phe Gln Asp Ser Ser Ser Gly Pro Cys Asp Thr Pro Ile Leu

115 120 125

Pro Gln

<210>47

<211>136

<212>PRT

<213>Artificial Sequence

<220>

<223> hCGbeta, delta126-135, S138C

<400>47

Ser Lys Glu Pro Leu Arg Pro Arg Cys Arg Pro Ile Asn Ala Thr Leu

1 5 10 15

Ala Val Glu Lys Glu Gly Cys Pro Val Cys Ile Thr Val Asn Thr Thr

20 25 30

Ile Cys Ala Gly Tyr Cys Pro Thr Met Thr Arg Val Leu Gln Gly Val

35 40 45

Leu Pro Ala Leu Pro Gln Val Val Cys Asn Tyr Arg Asp Val Arg Phe

50 55 60

Glu Ser Ile Arg Leu Pro Gly Cys Pro Arg Gly Val Asn Pro Val Val

65 70 75 80

Ser Tyr Ala Val Ala Leu Ser Cys Gln Cys Ala Leu Cys Arg Arg Ser

85 90 95

Thr Thr Asp Cys Gly Gly Pro Lys Asp His Pro Leu Thr Cys Asp Asp

100 105 110

Pro Arg Phe Gln Asp Ser Ser Ser Ser Ser Lys Ala Pro Pro Pro Gly Pro

115 120 125

Cys Asp Thr ProIle Leu Pro Gln

130 135

<210>48

<211>140

<212>PRT

<213>Artificial Sequence

<220>

<223> hCGbeta, delta131-135, S138C

<400>48

Ser Lys Glu Pro Leu Arg Pro Arg Cys Arg Pro Ile Asn Ala Thr Leu

1 5 10 15

Ala Val Glu Lys Glu Gly Cys Pro Val Cys Ile Thr Val Asn Thr Thr

20 25 30

Ile Cys Ala Gly Tyr Cys Pro Thr Met Thr Arg Val Leu Gln Gly Val

35 40 45

Leu Pro Ala Leu Pro Gln Val Val Cys Asn Tyr Arg Asp Val Arg Phe

50 55 60

Glu Ser Ile Arg Leu Pro Gly Cys Pro Arg Gly Val Asn Pro Val Val

65 70 75 80

Ser Tyr Ala Val Ala Leu Ser Cys Gln Cys Ala Leu Cys Arg Arg Ser

85 90 95

Thr Thr Asp Cys Gly Gly Pro Lys Asp His Pro Leu Thr Cys Asp Asp

100 105 110

Pro Arg Phe Gln Asp Ser Ser Ser Ser Ser Lys Ala Pro Pro Pro Ser Leu

115 120 125

Pro Ser Gly Pro Cys Asp Thr Pro Ile Leu Pro Gln

130 135

<210>49

<211>92

<212>PRT

<213>Artificial Sequence

<220>

<223>hCG alpha-subunit, Lys91 replaced with Glu

<400>49

Ala Pro Asp Val Gln Asp Cys Pro Glu Cys Thr Leu Gln Glu Asn Pro

1 5 10 15

Phe Phe Ser Gln Pro Gly Ala Pro Ile Leu Gln Cys Met Gly Cys Cys

20 25 30

Phe Ser Arg Ala Tyr Pro Thr Pro Leu Arg Ser Lys Lys Thr Met Leu

35 40 45

Val Gln Lys Asn Val Thr Ser Glu Ser Thr Cys Cys Val Ala Lys Ser

50 55 60

Tyr Asn Arg Val Thr Val Met Gly Gly Phe Lys Val Glu Asn His Thr

65 70 75 80

Ala Cys His Cys Ser Thr Cys Tyr Tyr His Glu Ser

85 90

<210>50

<211>92

<212>PRT

<213>Artificial Sequence

<220>

<223>hCG alpha-subunit loop 2, Lys91 replaced with Met

<400>50

Ala Pro Asp Val Gln Asp Cys Pro Glu Cys Thr Leu Gln Glu Asn Pro

1 5 10 15

Phe Phe Ser Gln Pro Gly Ala Pro Ile Leu Gln Cys Met Gly Cys Cys

20 25 30

Phe Ser Arg Ala Tyr Pro Thr Pro Leu Arg Ser Lys Lys Thr Met Leu

35 40 45

Val Gln Lys Asn Val Thr Ser Glu Ser Thr Cys Cys Val Ala Lys Ser

50 55 60

Tyr Asn Arg Val Thr Val Met Gly Gly Phe Lys Val Glu Asn His Thr

65 70 75 80

Ala Cys His Cys Ser Thr Cys Tyr Tyr His Met Ser

85 90

<210>51

<211>92

<212>PRT

<213>Artificial Sequence

<220>

<223>hCG alpha-subunit loop 2, Lys44 replaced with Ala

<400>51

Ala Pro Asp Val Gln Asp Cys Pro Glu Cys Thr Leu Gln Glu Asn Pro

1 5 10 15

Phe Phe Ser Gln Pro Gly Ala Pro Ile Leu Gln Cys Met Gly Cys Cys

20 25 30

Phe Ser Arg Ala Tyr Pro Thr Pro Leu Arg Ser Ala Lys Thr Met Leu

35 40 45

Val Gln Lys Asn Val Thr Ser Glu Ser Thr Cys Cys Val Ala Lys Ser

50 55 60

Tyr Asn Arg Val Thr Val Met Gly Gly Phe Lys Val Glu Asn His Thr

65 70 75 80

Ala Cys His Cys Ser Thr Cys Tyr Tyr His Lys Ser

85 90

<210>52

<211>92

<212>PRT

<213>Artificial Sequence

<220>

<223>hCG alpha-subunit loop 2, Lys44 replaced with Glu and Lys45 repla

ced with Gln

<400>52

Ala Pro Asp Val Gln Asp Cys Pro Glu Cys Thr Leu Gln Glu Asn Pro

1 5 10 15

Phe Phe Ser Gln Pro Gly Ala Pro Ile Leu Gln Cys Met Gly Cys Cys

20 25 30

Phe Ser Arg Ala Tyr Pro Thr Pro Leu Arg Ser Glu Gln Thr Met Leu

35 40 45

Val Gln Lys Asn Val Thr Ser Glu Ser Thr Cys Cys Val Ala Lys Ser

50 55 60

Tyr Asn Arg Val Thr Val Met Gly Gly Phe Lys Val Glu Asn His Thr

65 70 75 80

Ala Cys His Cys Ser Thr Cys Tyr Tyr His Lys Ser

85 90

<210>53

<211>92

<212>PRT

<213>Artificial Sequence

<220>

<223>hCG alpha-subunit loop 2, Lys44 replaced with Arg

<400>53

Ala Pro Asp Val Gln Asp Cys Pro Glu Cys Thr Leu Gln Glu Asn Pro

1 5 10 15

Phe Phe Ser Gln Pro Gly Ala Pro Ile Leu Gln Cys Met Gly Cys Cys

20 25 30

Phe Ser Arg Ala Tyr Pro Thr Pro Leu Arg Ser Arg Lys Thr Met Leu

35 40 45

Val Gln Lys Asn Val Thr Ser Glu Ser Thr Cys Cys Val Ala Lys Ser

50 55 60

Tyr Asn Arg Val Thr Val Met Gly Gly Phe Lys Val Glu Asn His Thr

65 70 75 80

Ala Cys His Cys Ser Thr Cys Tyr Tyr His Lys Ser

85 90

<210>54

<211>139

<212>PRT

<213>Artificial Sequence

<220>

<223>hCG analog-beta101-145, alpha, residues 3-100 deleted from hCG

 beta-subunit with alpha-subunit fused to the end of the remaining

beta-subunit

<400>54

Ser Lys Gly Gly Pro Lys Asp His Pro Leu Thr Cys Asp Asp Pro Arg

1 5 10 15

Phe Gln Asp Ser Ser Ser Ser Ser Lys Ala Pro Pro Pro Ser Leu Pro Ser

20 25 30

Pro Ser Arg Leu Pro Gly Pro Ser Asp Thr Pro Ile Leu Pro Gln Ala

35 40 45

Pro Asp Val Gln Asp Cys Pro Glu Cys Thr Leu Gln Glu Asn Pro Phe

50 55 60

Phe Ser Gln Pro Gly Ala Pro Ile Leu Gln Cys Met Gly Cys Cys Phe

65 70 75 80

Ser Arg Ala Tyr Pro Thr Pro Leu Arg Ser Lys Lys Thr Met Leu Val

85 90 95

Gln Lys Asn Val Thr Ser Glu Ser Thr Cys Cys Val Ala Lys Ser Tyr

100 105 110

Asn Arg Val Thr Val Met Gly Gly Phe Lys Val Glu Asn His Thr Ala

115 120 125

Cys His Cys Ser Thr Cys Tyr Tyr His Lys Ser

130 135

<210>55

<211>31

<212>PRT

<213>Homo sapiens

<400>55

Phe Gln Asp Ser Ser Ser Ser Ser Lys Ala Pro Pro Pro Ser Leu Pro Ser

1 5 10 15

Pro Ser Arg Leu Pro Gly Pro Ser Thr Asp Pro Ile Leu Pro Gly

20 25 30

<210>56

<211>10

<212>PRT

<213>Artificial Sequence

<220>

<223>Xl-Asp-Asp-Asp-Asp-Lys-Ser-Ym-Cys-Zn, where X, Y, and Z refer to

any tail portion amino acids and l, m, and n refer to the lengths

of the tail portion amino acids

<220>

<221>MISC_FEATURE

<223>xaa refers to any tail portion amino acids and n refers to the

lengths of the tail portion amino acids

<400>56

xaa _n Asp Asp Asp Asp Lys Ser xaa _n Cys xaa _n

1 5 10

<210>57

<211>92

<212>PRT

<213>Artificial Sequence

<220>

<223>An hCG truncated β-aubunit analog fused to the hCG alpha-carboxyterminus

<400>57

Ala Pro Asp Val Gln Asp Cys Pro Glu Cys Thr Leu Gln Glu Asn Pro

1 5 10 15

Phe Phe Ser Gln Pro Gly Ala Pro Ile Leu Gln Cys Met Gly Cys Cys

20 25 30

Phe Ser Arg Ala Tyr Pro Thr Pro Leu Arg Ser Lys Lys Thr Met Leu

35 40 45

Val Gln Lys Asn Val Thr Ser Glu Ser Thr Cys Cys Val Ala Lys Ser

50 55 60

Tyr Asn Arg Val Thr Val Met Gly Gly Phe Lys Val Glu Asn His Thr

65 70 75 80

Ala Cys His Cys Ser Thr Cys Tyr Tyr His Lys Ser Asp Asp Pro Arg

85 90 95

Phe Gly Pro Cys Asp Thr Pro Ile Leu Pro Gln

100 105

<210>58

<211>145

<212>PRT

<213>Artificial Sequence

<220>

<223>hCG beta-subunit with Cys substituted for Arg94

<400>58

Ser Lys Glu Pro Leu Arg Pro Arg Cys Arg Pro Ile Asn Ala Thr Leu

1 5 10 15

Ala Val Glu Lys Glu Gly Cys Pro Val Cys Ile Thr Val Asn Thr Thr

20 25 30

Ile Cys Ala Gly Tyr Cys Pro Thr Met Thr Arg Val Leu Gln Gly Val

35 40 45

Leu Pro Ala Leu Pro Gln Val Val Cys Asn Tyr Arg Asp Val Arg Phe

50 55 60

Glu Ser Ile Arg Leu Pro Gly Cys Pro Arg Gly Val Pro Asn Val Val

65 70 75 80

Ser Tyr Ala Val Ala Leu ser Cys Gln Cys Ala Leu Cys Cys Arg Ser

85 90 95

Thr Tbr Asp Cys Gly Gly Pro Lys Asp His Pro Leu Thr Cys Asp Asp

100 105 110

Pro Arg Phe Gln Asp Ser Ser Ser Ser Ser Lys Ala Pro Pro Pro Ser Leu

115 120 125

Pro Ser Pro Ser Arg Leu Pro Gly Pro Ser Asp Thr Pro Ile Leu Pro Gln

130 135 140

<210>59

<211>145

<212>PRT

<213>Artificial Sequence

<220>

<223>hCG beta-subunit with Cys substituted for Arg95

<400>59

Ser Lys Glu Pro Leu Arg Pro Arg Cys Arg Pro Ile Asn Ala Thr Leu

1 5 10 15

Ala Val Glu Lys Glu Gly Cys Pro Val Cys Ile Thr Val Asn Thr Thr

20 25 30

Ile Cys Ala Gly Tyr Cys Pro Thr Met Thr Arg Val Leu Gln Gly Val

35 40 45

Leu Pro Ala Leu Pro Gln Val Val Cys Asn Tyr Arg Asp Val Arg Phe

50 55 60

Glu Ser Ile Arg Leu Pro Gly Cys Pro Arg Gly Val Pro Asn Val Val

65 70 75 80

Ser Tyr Ala Val Ala Leu Ser Cys Gln Cys Ala Leu Cys Arg Cys Ser

85 90 95

Thr Thr Asp Cys Gly Gly Pro Lys Asp His Pro Leu Thr Cys Asp Asp

100 105 110

Pro Arg Phe Gln Asp Ser Ser Ser Ser Ser Lys Ala Pro Pro Pro Ser Leu

115 120 125

Pro Ser Pro Ser Arg Leu Pro Gly Pro Ser Asp Thr Pro Ile Leu Pro Gln

130 135 140

<210>60

<211>145

<212>PRT

<213>Artificial Sequence

<220>

<223>hCG beta-subunit with Cys substituted for Ser96

<400>60

Ser Lys Glu Pro Leu Arg Pro Arg Cys Arg Pro Ile Asn Ala Thr Leu

1 5 10 15

Ala Val Glu Lys Glu Gly Cys Pro Val Cys Ile Thr Val Asn Thr Thr

20 25 30

Ile Cys Ala Gly Tyr Cys Pro Thr Met Thr Arg Val Leu Gln Gly Val

35 40 45

Leu Pro Ala Leu Pro Gln Val Val Cys Asn Tyr Arg Asp Val Arg Phe

50 55 60

Glu Ser Ile Arg Leu Pro Gly Cys Pro Arg Gly Val Pro Asn Val Val

65 70 75 80

Ser Tyr Ala Val Ala Leu Ser Cys Gln Cys Ala Leu Cys Arg Arg Cys

85 90 95

Thr Thr Asp Cys Gly Gly Pro Lys Asp His Pro Leu Thr Cys Asp Asp

100 105 110

Pro Arg Phe Gln Asp Ser Ser Ser Ser Ser Lys Ala Pro Pro Pro Ser Leu

115 120 125

Pro Ser Pro Ser Arg Leu Pro Gly Pro Ser Asp Thr Pro Ile Leu Pro Gln

130 135 140

<210>61

<211>145

<212>PRT

<213>Artificial Sequence

<220>

<223>hCG beta-subunit with Cys substituted for Thr97

<400>61

Ser Lys Glu Pro Leu Arg Pro Arg Cys Arg Pro Ile Asn Ala Thr Leu

1 5 10 15

Ala Val Glu Lys Glu Gly Cys Pro Val Cys Ile Thr Val Asn Thr Thr

20 25 30

Ile Cys Ala Gly Tyr Cys Pro Thr Met Thr Arg Val Leu Gln Gly Val

35 40 45

Leu Pro Ala Leu Pro Gln Val Val Cys Asn Tyr Arg Asp Val Arg Phe

50 55 60

Glu Ser Ile Arg Leu Pro Gly Cys Pro Arg Gly Val Pro Asn Val Val

65 70 75 80

Ser Tyr Ala Val Ala Leu Ser Cys Gln Cys Ala Leu Cys Arg Arg Ser

85 90 95

Cys Thr Asp Cys Gly Gly Pro Lys Asp His Pro Leu Thr Cys Asp Asp

100 105 110

Pro Arg Phe Gln Asp Ser Ser Ser Ser Ser Lys Ala Pro Pro Pro Ser Leu

115 120 125

Pro Ser Pro Ser Arg Leu Pro Gly Pro Ser Asp Thr Pro Ile Leu Pro Gln

130 135 140

<210>62

<211>145

<212>PRT

<213>Artificial Sequence

<220>

<223>hCG beta-subunit with Cys substituted for Thr98

<400>62

Ser Lys Glu Pro Leu Arg Pro Arg Cys Arg Pro Ile Asn Ala Thr Leu

1 5 10 15

Ala Val Glu Lys Glu Gly Cys Pro Val Cys Ile Thr Val Asn Thr Thr

20 25 30

Ile Cys Ala Gly Tyr Cys Pro Thr Met Thr Arg Val Leu Gln Gly Val

35 40 45

Leu Pro Ala Leu Pro Gln Val Val Cys Asn Tyr Arg Asp Val Arg Phe

50 55 60

Glu Ser Ile Arg Leu Pro Gly Cys Pro Arg Gly Val Pro Asn Val Val

65 70 75 80

Ser Tyr Ala Val Ala Leu Ser Cys Gln Cys Ala Leu Cys Arg Arg Ser

85 90 95

Thr Cys Asp Cys Gly Gly Pro Lys Asp His Pro Leu Thr Cys Asp Asp

100 105 110

Pro Arg Phe Gln Asp Ser Ser Ser Ser Ser Lys Ala Pro Pro Pro Ser Leu

115 120 125

Pro Ser Pro Ser Arg Leu Pro Gly Pro Ser Asp Thr Pro Ile Leu Pro Gln

130 135 140

<210>63

<211>145

<212>PRT

<213>Artificial Sequence

<220>

<223>hCG beta-subunit with Cys substituted for Asp99

<400>63

Ser Lys Glu Pro Leu Arg Pro Arg Cys Arg Pro Ile Asn Ala Thr Leu

1 5 10 15

Ala Val Glu Lys Glu Gly Cys Pro Val Cys Ile Thr Val Asn Thr Thr

20 25 30

Ile Cys Ala Gly Tyr Cys Pro Thr Met Thr Arg Val Leu Gln Gly Val

35 40 45

Leu Pro Ala Leu Pro Gln Val Val Cys Asn Tyr Arg Asp Val Arg Phe

50 55 60

Glu Ser Ile Arg Leu Pro Gly Cys Pro Arg Gly Val Pro Asn Val Val

65 70 75 80

Ser Tyr Ala Val Ala Leu Ser Cys Gln Cys Ala Leu Cys Arg Arg Ser

85 90 95

Thr Thr Cys Cys Gly Gly Pro Lys Asp His Pro Leu Thr Cys Asp Asp

100 105 110

Pro Arg Phe Gln Asp Ser Ser Ser Ser Ser Lys Ala Pro Pro Pro Ser Leu

115 120 125

Pro Ser Pro Ser Arg Leu Pro Gly Pro Ser Asp Thr Pro Ile Leu Pro Gln

130 135 140

<210>64

<211>95

<212>PRT

<213>Artificial Sequence

<220>

<223>An hCG alpha-subunit analog with Gly-Gly-cys at its carboxyterminus

<400>64

Ala Pro Asp Val Gln Asp Cys Pro Glu Cys Thr Leu Gln Glu Asn Pro

1 5 10 15

Phe Phe Ser Gln Pro Gly Ala Pro Ile Leu Gln Cys Met Gly Cys Cys

20 25 30

Phe Ser Arg Ala Tyr Pro Thr Pro Leu Arg Ser Lys Lys Thr Met Leu

35 40 45

Val Gln Lys Asn Val Thr Ser Glu Ser Thr Cys Cys Val Ala Lys Ser

50 55 60

Tyr Asn Arg Val Thr Val Met Gly Gly Phe lys Val Glu Asn His Thr

65 70 75 80

Ala Cys His Cys Ser Thr Cys Tyr Tyr His Lys Ser Gly Gly Cys

86 90 95

<210>65

<211>92

<212>PRT

<213>Artificial Sequence

<220>

<223>An hCG alpha-subunit analog with Asp in place of Asn52 and Cys in place of

Ser92

<400>65

Ala Pro Asp Val Gln Asp Cys Pro Glu Cys Thr Leu Gln Glu Asn Pro

1 5 10 15

Phe Phe Ser Gln Pro Gly Ala Pro Ile Leu Gln Cys Met Gly Cys Cys

20 25 30

Phe Ser Arg Ala Tyr Pro Thr Pro Leu Arg Ser Lys Lys Thr Met Leu

35 40 45

Val Gln Lys Asp Val Thr Ser Glu Ser Thr Cys Cys Val Ala Lys Ser

50 55 60

Tyr Asn Arg Val Thr Val Met Gly Gly Phe Lys Val Glu Asn His Thr

65 70 75 80

Ala Cys His Cys Ser Thr Cys Tyr Tyr His Lys Ser

85 90

<210>66

<211>145

<212>PRT

<213>Artificial Sequence

<220>

<223>hCG beta-subunit with Cys substituted for Ser96 and hFSH beta-subunit

residues 95-108 for hCG beta-subunit residues 101-108

<400>66

Ser Lys Glu Pro Leu Arg Pro Arg Cys Arg Pro Ile Asn Ala Thr Leu

1 5 10 15

Ala Val Glu Lys Glu Gly Cys Pro Val Cys Ile Thr Val Asn Thr Thr

20 25 30

Ile Cys Ala Gly Tyr Cys Pro Thr Met Thr Arg Val Leu Gln Gly Val

35 40 45

Leu Pro Ala Leu Pro Gln Val Val Cys Asn Tyr Arg Asp Val Arg Phe

50 55 60

Glu Ser Ile Arg Leu Pro Gly Cys Pro Arg Gly Val Pro Asn Val Val

65 70 75 80

Ser Tyr Ala Val Ala Leu Ser Cys Gln Cys Ala Leu Cys Arg Arg Cys

85 90 95

Thr Thr Asp Cys Thr Val Arg Gly Leu Gly Pro Ser Tyr Cys Ser Phe

100 105 110

Gly Glu Phe Gln Asp Ser Ser Ser Ser Ser Lys Ala Pro Pro Pro Ser Leu

115 120 125

Pro Ser Pro Ser Arg Leu Pro Gly Pro Ser Asp Thr Pro Ile Leu Pro Gln

130 135 140

Claims (19)

1. composition comprises:

Protein portion, wherein this protein portion contains the alternate cysteine residues in the need mark;

Be positioned at the afterbody of protein portion end; And

Tubercle, wherein this tubercle is positioned at the free-end of afterbody, and contains cysteine residues, and nodular cysteine residues can form disulfide linkage with the alternate halfcystine of protein portion.

2. the composition of claim 1, wherein afterbody contains proteolytic enzyme cutting site.

3. the composition of claim 1, wherein tubercle comprises phenotypic markers.

4. the composition of claim 1, wherein tubercle comprises polypeptide.

5. the composition of claim 1, wherein tubercle comprises protein.

6. the composition of claim 5, wherein nodular halfcystine is positioned at proteinic surface.

7. the composition of claim 1, wherein protein portion is a monomeric protein.

8. the composition of claim 1, wherein protein portion is a polymer protein.

9. method at the specific site labelled protein comprises:

A) select desired protein;

B) want the mark part to position to desired protein;

C) select required tubercle, wherein required tubercle contains cysteine residues;

D) preparation coding desired protein, afterbody and required nodular construct, wherein desired protein is wanting the mark part to have alternate half Guang amino acid;

E) construct is imported in a kind of cell protein with presentation markup, wherein halfcystine in the tubercle and the alternate halfcystine in the desired protein form disulfide linkage.

10. the method in the claim 9, wherein tubercle comprises phenotypic markers, signal sequence or protein.

11. the method in the claim 10, wherein tubercle protein is proteolytic enzyme.

12. the method in the claim 9, wherein desired protein exists with monomeric form.

13. the method in the claim 9, wherein desired protein exists with the polymer form.

14. a method for purifying proteins comprises:

A) with effable construct transfered cell, this construct a kind of protein of encoding wherein, this protein is wanting the mark part to comprise the alternate halfcystine, comprise afterbody at proteinic end, this afterbody contains the cleavage site of proteolytic enzyme, at the terminal nodosity of afterbody, this tubercle contains cysteine residues;

B) lysing cell;

C) according to nodular feature protein purification.

15. the method for a mark hCG comprises:

A) preparation can be expressed the construct of natural hCG β or hCG β-S138C;

B) preparation can be expressed the construct of natural hCG α or the alternative analogue of hCG α halfcystine;

C) with step a) and b) construct of preparation imports in the COS-7 cell and carries out coexpression.

16. the method for claim 15, wherein the construct of step a) also comprises protein is merged mutually with the 140th or 145 residue of hCG β.

17. the method for claim 16, wherein protein is β-Nei Xiananmei.

18. the method for claim 15, wherein hCG α halfcystine substitutes analogue and is selected from SEQ ID NO:1 to SEQ ID NO:35.

19. a method of surveying and drawing distance between protein molecule comprises:

A) select first protein molecule;

B) select second protein molecule, wherein first protein molecule and second proteins interaction;

C) generate first protein molecule, every kind first protein molecule that is wherein produced contains a tubercle on the first proteinic different loci;

D) generate second protein molecule;

E) protein analysis first protein that utilizes step c) and step d) and generated and the distance between second protein.

Images