JP2001509390A - Fusion protein containing coiled coil structure - Google Patents

Abstract

  (57) [Summary]   The present invention relates to a fusion protein comprising: a) a first region capable of forming a coiled-coil structure; and b) a second region containing a polypeptide of interest and not naturally related to the first region. The fusion proteins of the invention are applicable to the production of very small polypeptides in transformed host cells.

Description

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to novel fusion partners for recombinantly produced proteins. In particular, the present invention relates to polypeptides having a coiled-coil structure that is resistant to proteolysis and that has the ability to act as a carrier and increase the immunogenicity of the protein fused thereto.

[0002] Recombinant DNA technology has enabled the industry to produce many commercially important proteins. Proteins are produced in a wide range of expression systems based on, for example, bacteria, yeast, insect, plant and mammalian cells. One of the problems associated with recombinant protein production is that host cells contain enzymes that degrade the protein, and the presence of such enzymes makes production of small polypeptides particularly difficult. .

[0003] One approach to overcoming such difficulties is to express the recombinant protein of interest in the form of a fusion protein. The DNA encoding the protein of interest is fused in frame with the fusion partner protein, and the resulting fusion is expressed. In many cases, by including a linker sequence that encodes a protease cleavage located between the two components of the fusion, the fusion can be cleaved after recovery from the host cell.

[0004] Fusion partner proteins are often those that can be recovered and purified by some form of highly specific affinity purification method. Such proteins include, for example, glutathione-S-transferase, maltose binding protein and β-lactamase.

[0005] However, all of these fusion partner proteins have some drawbacks because they are relatively large. For example, their fusion partner proteins are too large to allow the protein of interest to function with some degree of independence, so it is essential to remove them before any meaningful manipulation can be performed on the protein of interest. It is. Thus, many small polypeptides are still produced by chemical synthesis.

[0006] The present invention, the present inventors have cattle ATP-ase inhibitor protein, which was made while investigating the structure of IF _1. It is a small, or 84 amino acid, protein that helps regulate ATP synthase activity in mitochondria. When the present inventors analyzed this protein, it was found that the C-terminal region (amino acids 48 to 84) of the protein formed a coiled coil structure.

[0007] Coiled coils are formed by two or three α-helices that are parallel and properly aligned and exhibit a pattern of hydrophilic and hydrophobic residues that repeat every seven residues (Lupas et al., Science). 252; 1162-1164, 1991). The right-handed α-helix wraps around each other, forming a slightly left-handed superhelical twist. Coiled coils are well known in fibrous proteins such as keratin, myosin and tropomyosin, but are also found in other proteins, such as proteins containing leucine zipper motifs.

[0008] The coiled-coil region of a protein generally contains seven residue repeats, with the amino acids at each repeat indicated by ag. Amino acids a and d in each repeat are generally hydrophobic amino acids. Several algorithms are available for predicting the presence of coiled coils in proteins. See, e.g., Lupas et al., 1991 and Berger et al., Proc. Nat.
l. Acad. Sci. (USA) 92; 8259-8263, 1995. The latter is considered to be more stringent in detecting double-stranded coils, but less efficient in detecting triple-stranded coils.

[0009] The present inventors, E.Col several fusion proteins containing short sequence of coiled-coil region and a second protein bovine IF ₁ ATP-ase inhibitor protein
i. The present inventors have found that these fusion proteins capable of efficiently recovering the second protein can be expressed at a high level and isolated as they are. Thus, by using coiled-coil as a fusion partner protein in a recombinant expression system, it is possible to express the desired polypeptide in a manner that protects the polypeptide from degradation in host cells. Accordingly, the present invention provides a fusion protein comprising: a) a first region capable of forming a coiled coil structure; and b) a second region containing a polypeptide sequence of interest and not naturally associated with the first region. .

[0010] The first region may be located at the N-terminus or C-terminus of the second region. However, the first region is preferably located at the N-terminus of the second region, most preferably at the N-terminus of the fusion protein. Preferably, the fusion protein further comprises a cleavable linker region between the first region and the second region. The invention further includes a nucleic acid encoding a fusion protein of the invention, which nucleic acid preferably forms part of an expression vector comprising the nucleic acid operably linked to a promoter.

[0011] The present invention further provides a host cell having the expression vector of the present invention, and (i) culturing the host cell under conditions that cause expression of the fusion protein from the expression vector in the host cell; and (ii) ) A method for preparing the fusion protein of the present invention, including recovering the fusion protein from cells. If the fusion protein further comprises a protease-cleavable linker region between the first and second regions, the method optionally comprises cleaving the protein with a protease-cleavable linker portion, and recovering the second region. Further comprising:

[0012] BRIEF DESCRIPTION OF THE DRAWINGS Figure 1: E.coli C41 (DE3) were expressed in cells, UncI and various fragments fused to truncations of uncII gene (truncated The) Expression plug encoding IF ₁ inhibitor plasmid, and Products separated on Coomassie stained SDS-PAGE gel. Lanes are as _{follows: 1: IF 1; 2:} IF 1 -a1; 3: IF 1 -a3; 4: IF 1 -I1; 5: IF1-I2
. The molecular weight markers to the left; a is subunit a of E.coli F ₁ F ₀ ATP synthase; I is a subunit I of E.coli F ₁ F ₀ ATP synthase. See Table 1 for the complete nomenclature.

FIG. 2: A peptide of 2 to 54 amino acids in length separated on SDS-PAGE, IF ₁ (44
~ 84) as a fusion. See detailed description.

FIG. 3: Purification of the fusion protein by reverse phase HPLC. A and C: IF ₁ -a1 and
Chromatogram of the purified product of IF ₁ -a3. B and D: SDS-PAGE analysis of purified IF ₁ -a1 and IF ₁ -a3, respectively. IB: inclusion body; numbers indicate collected fractions.

FIG. 4: Purification of the fusion protein by reverse phase HPLC. A and C: IF ₁ -I1 and
Chromatogram of the purified product of IF ₁ -I2. B and D: SDS-PAGE analysis of each refining of IF ₁ -I1 and IF ₁ -I2. IB: inclusion body; numbers indicate collected fractions.

FIG. 5: Purification of the fusion protein of the invention by other methods. 1 /: Inclusion body origin:
If-a1 is expressed as inclusion bodies and solubilized using 6M guanidinium chloride.
Purify by reverse phase HPLC as described. The If-UCP270 fusion protein is solubilized in the presence of urea and purified on a nickel column in the presence of LDAO detergent (see text for details). 2 /: Three fusion proteins, If-UCP1P1, If-
UCP2P2 and If-UCP3P3 are solubilized in bacterial cytoplasm and purified by anion-exchange chromatography on S-Sepharose followed by affinity chromatography on a nickel column. After purification of all protein samples,
Analyze by PAGE. The gel is stained with Coomassie blue dye 83.

[0017]Detailed description of the invention A: First Region The first region of the fusion protein of the present invention may include any natural or synthetic coiled-coil polypeptide. Such regions include keratin, myosin, tropomyosin
Or various proteins such as laminin, or GCN4 or hemagglutinin
Protein, such as a protein containing a leucine zipper motif, or IF ₁ ATPase inhibitor protein, preferably mammalian IF₁ ATPaseinhi
Found in other proteins such as bitter proteins. Rat and bovine
The sequence of the protein is described in the examples. Human or mouse IF₁ Other mammalian IFs such as ATPase inhibitor protein₁ ATPase inhibitor proteins may be used.

It is not necessary to use the entire protein in which the coiled-coil region is found. In the case of a large coiled coil protein having a large number of 7-residue repeats capable of forming a coiled coil structure, it is not always necessary to use the entire coiled coil region. Portions of that area can be used subject to size and functional limitations discussed below.

In addition, synthetic variants of such proteins or regions thereof can be used, provided that they maintain the ability to form coiled coils. Since the region of the coiled-coil protein that forms the coiled-coil has a heptad repeat structure, synthetic variants may be substituted for the native protein by substitution, particularly conservative substitutions, or by insertion of one or more full heptad repeats. Are generally different.

When conservative substitutions are made, they can be made with reference to the following table, wherein the amino acids in the same block in the second column, preferably in the same row in the third column, can be substituted for each other. When one or more heptad repeats are inserted into the structure of a native coiled coil protein, the insertion is made to maintain the ag residue structure described above. Conveniently, the insertion of one or more complete ag repeats is between g and a of successive repeats of the native protein. However, insertions can also be made between other residues of the heptad repeat as long as the insert maintains the heptad structure.

[0021] Synthetic variants of the native coiled-coil protein can be produced by standard recombinant DNA techniques. For example, by using site-directed mutagenesis, changes can be made in the coding region of the DNA encoding the native coiled-coil protein. If an insertion is made, the synthetic DNA encoding the insert and the 5 'and 3' flanking regions corresponding to the native sequence are inserted on either side of the insertion site. The flanking regions contain convenient restriction sites that correspond to those in the native sequence so that the sequence can be cut with the appropriate enzymes and synthetic DNA ligated into the cut. The DNA is then expressed according to the invention to produce the encoded protein. These methods are merely illustrative of the many standard techniques known in the art for manipulating DNA sequences, and other known techniques may be used.

[0022] The fusion proteins of the invention contain the required number of 7 residue repeats to form a coiled coil structure. Generally, it will be three or more (smaller is better to minimize the contribution of the coiled-coil forming sequence to the fusion protein). This can be particularly important where it is often necessary to perform radioisotope labeling with ¹⁵ N or ¹³ C in determining the structure of the protein by NMR. This is expensive and for long fusion partners, much of the incorporated radioactivity is removed when the carrier protein (eg, often GST) is cleaved. Thus, the fusion partner is about 25-100 amino acids in size, containing 3-14, preferably 4-10, 7-residue repeat units capable of forming a coiled-coil structure.

The ability of a natural or synthetic coiled-coil sequence to form a coiled coil can be tested by any of the methods described in the examples known in the art.

[0024] The sequence of any putative coiled-coil region can be investigated by the algorithm of Lupas et al. Or Berger et al. Sequences can also be synthesized by recombinant DNA technology, and solutions of the sequences can be tested for circular dichroism spectra at various concentrations. When coiled-coil formation occurs (as opposed to non-specific aggregation), the spectrum is constant over a range of dilutions (while non-specific aggregation is diluted).

The first region may also include sequences flanking the seven residue repeat. These may need to maintain the overall structure of the first region such that a heptad repeat α-helical coil structure can be formed. For example, four heptad repeats of bovine IF ₁ ATP-ase inhibitors are found in residues 53-80. We believe that residues 44-84 of this protein
Has been found to be a preferred embodiment of the present invention. However, other suitable regions of the protein or corresponding portions of a mammalian homolog, such as 1-84, such as 23-84, or 1-78, such as 44-78, may be used.

Thus, generally, from 0 to 100, such as from 5 to 50, non-coiled, coil-forming amino acids may be present at either or both the N- and / or C-terminus of the first region. These sequences may be from the protein from which the coiled-coil region is derived or from any other natural or synthetic source. In principle, this area is not important if it does not affect the formation of the coiled coil structure.

B: Second Region The second region of the fusion protein of the invention may comprise any polypeptide sequence of interest unrelated to the first region in nature. Usually, this means that the sequence of interest is known to be encoded by a gene different from the gene that encodes the first region in nature. This can be easily confirmed by examining the sequence of the first and second regions against a publicly available sequence data bank. The second region can be of the same or different species as the first region. The first and second regions are derived from portions of the same protein, but can be present in the fusion protein of the invention in a manner different from the native protein sequence.

Although the fusion proteins of the invention can be of any size, generally, the invention provides that the polypeptide sequence of interest is short, eg, 2-100 amino acids in length, preferably 2-5 amino acids in length.
It is particularly useful when there are 0 amino acids, or 2 to 30 or 5 to 10 amino acids. However, large polypeptide sequences, e.g. up to 150, 200, 400 or 100
Up to zero amino acids are also contemplated. The present invention is particularly advantageous for the preparation of small polypeptides that are currently difficult to produce by recombinant methods. Such polypeptides include, for example, chaperone proteins, metabolic enzymes, DNA and RNA binding proteins, antibodies, viral proteins, intrinsic membrane proteins (mitochondrial-derived transport proteins, 7-helix receptor molecules, T-cell receptor And small molecules derived from multisubunit biological structures such as cytoskeletal complexes, antibody binding peptides, peptide hormones (and other bioactive peptides produced by ribosome synthesis), and respiratory enzymes ATP synthase Subunits. In general, the invention is suitable for use with peptides of any dimension, but its advantageous properties include small polypeptides, e.g.
It is best utilized with amino acids, especially 2-20 amino acids in length, preferably 5-10 amino acids in length.

A particular advantage of the present invention is that peptides which were so short that they were previously produced by oligopeptide synthesis technology can be produced by recombinant DNA technology. Thus, it is possible to produce a library of peptides, for example mutants of bioactive peptides, which can be screened inexpensively and efficiently in recombinant expression systems, especially bacterial expression systems, or otherwise analyzed. Can be done.

C: cleavable linker region When the first and second regions are linked by a cleavable linker region, this may be a region suitable for this purpose. Preferably, the cleavable linker region is a protease cleavable linker, but other linkers cleavable, for example, by small molecules may be used. They include Me that can be cut by cyanogen bromide.
tX site, Asn-Gly cleavable by hydroxylamine, cleavable by weak acid
Includes Asp-Pro, and in particular Trp-X, which is cleavable by NBS-skatole. Protease cleavage sites are preferred because cleavage conditions are milder, such as are found in factor Xa, thrombin and collagenase. Any of these can be used. The exact sequence is available in the art and one of skill in the art would not have difficulty choosing an appropriate cleavage site. For example, the protease cleavage region targeted by factor Xa is IEGR. The protease cleavage region targeted by enterokinase is DDDDK. The protease cleavage region targeted by thrombin is LVPRG.

D: Nucleic Acid The present invention also provides a nucleic acid encoding the fusion protein of the present invention. These can be constructed using standard recombinant DNA techniques. The nucleic acid can be RNA or DNA, and is preferably DNA. If the nucleic acid is RNA, the manipulation can be performed with a cDNA intermediate. Generally, a nucleic acid sequence encoding the first region is prepared, and appropriate restriction sites are provided at the 5 'and / or 3' end. Conveniently, the array is
Operate on a standard laboratory vector, such as a plasmid vector based on pBR322 or pUC19 (see below). Molecular Cloning (Col. by Sambrook et al.)
d Spring Harbor, 1989) or a similar standard reference which details the appropriate technique.

[0032] The nucleic acid encoding the second region can be similarly provided in a similar vector system. The origin of a nucleic acid can be ascertained by reference to a publication or a data bank, such as a gene bank.

[0033] The nucleic acid encoding the desired first or second region may be obtained from an academic or commercial source, provided that only such sequence data is available. It is willing to supply raw materials by synthesizing or cloning the appropriate sequence. Generally, this can be done by reference to literature describing the cloning of the gene in question. Also, when available sequence data is limited, or when it is desirable to express a nucleic acid homologous or related to a known nucleic acid, a representative nucleic acid is a nucleotide sequence that hybridizes to a nucleic acid sequence known in the art. Can be characterized as

[0034] The stringency of hybridization refers to conditions under which a polynucleic acid hybrid is stable. Such conditions will be clear to the skilled person. As known to those skilled in the art, the stability of a hybrid is determined by the melting temperature (Tm) of the hybrid.
Approximately 1-1.5 ° C. decreases for every 1% decrease in sequence homology. In general, the stability of a hybrid is a function of sodium ion concentration and temperature. Typically, hybridization reactions are performed under conditions of high stringency, followed by various stringency washes.

As used herein, high stringency means conditions that allow hybridization of only nucleic acid sequences that form stable hybrids at 1 M Na +, 65-68 ° C. High stringency conditions include, for example, an aqueous solution containing 6 × SSC, 5 × Denhardt, 1% SDS (sodium dodecyl sulfate), 0.1Na + pyrophosphate and 0.1 mg / ml denatured salmon sperm DNA as a non-specific competitor. Can be obtained by hybridization in After hybridization,
Several steps of high stringency washing can be performed by final washing (at about 30 minutes) in the hybridization temperature in 0.2-0.1 x SSC, 0.1% SDS.

Medium stringency refers to conditions equivalent to hybridization in the above solutions except at about 60-62 ° C. In this case, the final wash is 1 × SSC, 0.1
Performed at hybridization temperature in% SDS. Low stringency refers to conditions equivalent to hybridization in the above solution at about 50-52 ° C. In this case, the final wash is performed at the hybridization temperature in 2 × SSC, 0.1% SDS.

It is understood that these conditions can be adjusted and reproduced using various buffers, such as formamide based buffers, and various temperatures.
Denhardt's solution and SSC can be prepared using other suitable hybridization buffers (for example, edited by Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, Cold S.
pring Harbor Laboratory Press, New York or Ausubel et al. (1990) Curr
ent Protocols in Molecular Biology, John Wiley & Sons, Inc.). Optimal hybridization conditions depend on probe length and
GC content also plays a role and must be determined empirically.

[0038] Given the guidance herein, nucleic acids suitable for forming the first or second regions of the fusion proteins of the invention can be obtained according to methods well known in the art. It is. For example, the DNA of the present invention may be a genomic library prepared by chemical synthesis using the polymerase chain reaction (PCR) or from a source that contains the desired nucleic acid and that will express it at a detectable level. Alternatively, it can be obtained by screening an appropriate cDNA library.

Methods for the chemical synthesis of nucleic acids of interest are known in the art and include the triester, phosphite, phosphoramidite and H-phosphonate methods, PCR and other self-priming methods and solid-state methods. Oligonucleotide synthesis on a carrier. These methods can be used when the entire nucleic acid sequence of the nucleic acid is known, or when the sequence of the nucleic acid complementary to the coding strand is available. Also, if the target amino acid is known, a possible nucleic acid sequence can be deduced using known and preferred coding residues for each amino acid residue.

Another means for isolating the gene encoding the desired region of the fusion protein is to use the PCR technique, for example, as described in Section 14 of Sambrook et al., 1989. This method requires the use of an oligonucleotide probe that hybridizes to the desired nucleic acid. The strategy for oligonucleotide selection is shown below.

[0041] The library is screened using probes or analytical tools designed to identify the gene of interest or the protein encoded thereby. Suitable means for a cDNA expression library include recognizing the desired protein,
A monoclonal or polyclonal antibody that specifically binds thereto; known or suspected to encode a desired protein from the same or a different species
Oligonucleotides that are about 20-80 in length encoding cDNAs; and / or complementary or homologous cDNAs or fragments thereof encoding the same or hybridizing genes. Suitable probes for screening genomic DNA libraries include, but are not limited to, cDNAs or fragments thereof encoding the same or hybridizing DNA; and / or homologous genomic cDNAs or fragments thereof.

The nucleic acid encoding the desired protein can be isolated by screening a suitable cDNA or genomic library with the probe under suitable hybridization conditions.

As used herein, a probe is, for example, identical (or its complement) to an equal or greater number of contiguous bases from a known or desired sequence.
It is a single-stranded DNA or RNA having a sequence of nucleotides containing 10 to 50, preferably 15 to 30, most preferably at least about 20 contiguous bases. The nucleic acid sequence selected as a probe should be of sufficient length and sufficiently distinct to minimize false positive results. The nucleotide sequence is usually based on a conserved or highly homologous nucleotide sequence or region of the desired protein. Nucleic acids used as probes are those that can degenerate at one or more positions. The use of degenerate oligonucleotides can be particularly important when screening libraries from a species where the preferential codon usage in that species is unknown.

Preferred regions for constructing probes include 5 ′ and / or 3 ′ coding sequences, sequences predicted to encode a ligand binding site, and the like. For example, the full-length cDNA clone disclosed herein as SEQ ID NO: 1 or a fragment thereof is:
In particular, it can be used as a probe for isolating the gene encoding the first region. Preferably, the nucleic acid probe of the present invention is labeled with a suitable labeling means for easily detecting hybridization. For example, a suitable labeling means is a radioactive label. A preferred method of labeling DNA fragments is by mixing α ³² P dATP with a Klenow fragment of DNA polymerase in a random priming reaction, as is well known in the art. Oligonucleotides are usually γ ³² P-labeled
End-labeled with ATP and polynucleotide kinase. However, fragments or oligonucleotides can also be labeled using other methods (eg, non-radioactive), such as enzyme labeling, fluorescent labeling with a suitable fluorophore, and biotinylation.

[0045] The library may be, for example, a portion of DNA containing substantially the entire desired sequence or the DNA.
After screening with the appropriate oligonucleotides based on a portion of the DNA, positive clones are identified by detecting the hybridization signal, and the identified clones are characterized by restriction enzyme mapping and / or DNA sequence analysis, Check to see if they contain the DNA encoding the complete polypeptide (ie, whether they contain translation initiation and termination codons). If the selected clones are incomplete, they can be used to rescreen the same or a different library to obtain overlapping clones. If the library is a genomic library, the overlapping clones can include exons and introns. If the library is a cDNA library, the overlapping clone contains an open reading frame.
In each case, complete clones can be identified by comparison to the DNA and deduced amino acid sequences provided herein.

It is contemplated that the nucleic acids of the present invention can be readily modified by nucleotide substitution, nucleotide deletion, nucleotide insertion or inversion of the direction of nucleotide extension, and any combination thereof. By using such a mutant, for example, a mutant having an amino acid sequence different from the sequence found in nature can be produced. Mutagenesis can be predetermined (can be site-specific) or random. Mutations that are not silent mutations should not place sequences from the reading frame and preferably do not create complementary regions that can hybridize to give rise to secondary mRNA structures such as loops or hairpins.

The foregoing methods can, of course, be applied to the identification and modification or generation of useful sequences in any part of the fusion proteins of the invention. In particular, SEQ ID NO: 1 herein
Its suitable fragments such as sequence or the aforementioned IF ₁ polypeptide coiled-coil shown as may be used as probes to further identify other suitable arrangement.

The first or second region may be manipulated to introduce an appropriate restriction enzyme site at its end, so that the first or second region is linked to the nucleic acid encoding the first region via the corresponding restriction enzyme site. Become. Desirably, such sites are identical or at least have compatible cohesive ends. Of course, the first and second regions can be joined by other means, for example, the first region can be incorporated into a primer used to isolate or replicate the second region.

If a protease cleavable linker region is required, such regions may be introduced into the linked first and second regions (eg, a restriction site linking the two) or may be introduced prior to their joining. To one or the other.

E. Expression Vectors and Host Cells The nucleic acids encoding the fusion proteins of the invention, or components thereof, can be incorporated into vectors for subsequent manipulation. As used herein, a vector (or plasmid) is intended to mean a discrete element used to introduce heterologous DNA into a cell for its expression or replication. The selection and use of such mediators is well within the skill of those in the art. Many vectors are available and the selection of the appropriate vector will determine the intended use of the vector, i.e., whether it will be used for DNA amplification or whether it will be used for DNA expression. It depends on the size of the DNA as well as the host cell transformed with the vector. Each vector contains various components depending on its function (amplification of the DNA or expression of the DNA) and the host cell with which it is compatible. Vector components generally include, but are not limited to, one or more of an origin of replication, one or more marker genes, an enhancer element, a promoter, a transcription termination sequence, and a signal sequence.

[0051] Both expression and cloning vectors generally contain a nucleic acid sequence that enables the vector to replicate in one or more selected host cells. Typically, in cloning vectors, these sequences are those that allow the vector to replicate independently of the host chromosomal DNA and include origins of replication or autonomously replicating sequences.
Such sequences are well known for various bacteria, yeasts and viruses. The origin of replication from plasmid pBR322 is suitable for most Gram-negative bacteria, the 2μ plasmid origin is suitable for yeast, and various viral origins (eg, SV40, polyoma, adenovirus) are used for cloning vectors in mammalian cells. Useful for Generally, origins of replication components are not required in mammalian expression vectors unless they are used for high levels of DNA replication in mammalian cells, such as COS cells.

[0052] Most expression vectors are shuttle vectors, ie, they are replication competent in at least one class of organisms, but can be transfected into another organism for expression. For example, a vector is cloned into E. coli and then transfected into yeast or mammalian cells, even if the vector does not have the ability to replicate independently of the host cell chromosome. DNA can also be replicated by insertion into the host genome. However, the recovery of genomic DNA encoding the fusion protein of the present invention is more complicated than the recovery of exogenously replicated vectors, since excision of the DNA requires restriction enzyme digestion. DNA can be amplified by PCR and transfected directly into host cells without any replication components.

[0053] Conveniently, the expression and cloning vectors may contain a selection gene, also termed a selectable marker. This gene encodes a protein necessary for the survival or growth of transformed host cells grown on the selection medium. Host cells not transformed with the vector containing the selection gene will not survive in the medium. Typical selection genes are antibodies and other toxins such as proteins that are resistant to ampicillin, neomycin, methotrexate or tetracycline, supplement a complement auxotrophy, or supply important nutrients that are not available from complex media. Code.

With respect to selection gene markers suitable for yeast, any marker gene that facilitates the selection of transformants by phenotypic expression of the marker gene can be used. Suitable markers for yeast are, for example, those that have resistance to the antibiotic G418, hygromycin or bleomycin, or confer prototrophy in an auxotrophic yeast mutant such as the URA3, LEU2, LYS2, TRP1, or HIS3 gene .

Since the replication of the vector is conveniently made in E. coli, an E. coli gene marker and an E. coli origin of replication are conveniently included. These are pBR322, Bluesc
ript (copyright) vector or a pUC plasmid obtained from an E. coli plasmid containing both an E. coli origin of replication, such as pUC18 or pUC19, and an E. coli gene marker that is resistant to antibiotics such as ampicillin. obtain.

Suitable selectable markers for mammalian cells include identifying cells capable of taking up vector nucleic acids, such as dihydrofolate reductase (DHFR, methotrexate resistance), thymidine kinase, or genes with resistance to G418 or hygromycin. Is what makes it possible. Mammalian cell transformants are placed under selection pressure so that only transformants that have taken up and expressing the marker are the only survivors suitable. In the case of a DHFR or glutamine synthase (GS) marker, the selection pressure is determined by culturing the transformants under conditions of increasing pressure, whereby both the selection gene and the ligated DNA encoding the fusion protein (in its chromosome) By causing amplification (at the integration site). Amplification is a process in which genes that are in great demand for the production of proteins essential for growth, as well as closely related genes that can encode the desired protein, are repeated vertically in the chromosome of the recombinant cell. Large amounts of the desired protein are usually synthesized from the amplified DNA.

Expression and cloning vectors usually contain a promoter that is recognized by the host organism and is operably linked to the fusion protein-encoding nucleic acid. Such a promoter can be inducible or constitutive. The promoter is operably linked to DNA encoding the fusion protein by removing the promoter from the source DNA by restriction enzyme digestion and inserting the isolated promoter sequence into the vector. The use of a native promoter sequence, one of the components of the fusion protein, and many heterologous promoters can induce DNA amplification and / or expression. The term “operably linked” means that the components described are juxtaposed in a relationship permitting them to function in their intended manner. A regulatory sequence "operably linked" to a coding sequence is ligated such that expression of the coding sequence is obtained under conditions compatible with the regulatory sequences.

Suitable promoters for use in prokaryotic hosts include, for example, β-lactamase and lactose promoter systems, alkaline phosphatase, tryptophan (trp) promoter systems, and hybrid promoters such as the tac promoter. Their nucleotide sequences are published, so that those skilled in the art can functionally ligate to them the DNA encoding the fusion protein using linkers or adapters to provide any necessary restriction sites. Promoters for use in bacterial systems also generally contain a Shine-Dalgarno sequence operably linked to the DNA encoding the fusion protein.

Preferred expression vectors are bacterial expression vectors that include a bacteriophage promoter, such as phagex or T7, that has the ability to function in bacteria. In one of the most widely used expression systems, the nucleic acid encoding the fusion protein can be transcribed from the vector by T7 RNA polymerase (Studier et al., Methods
in Enzymol. 185; 60-89, 1990). E. coli BL2 used with pET vector
In the 1 (DE3) host strain, T7 RNA polymerase is a λ-lysogen in the host bacterium.
Produced from (lysogen) DE3, its expression is under the control of the IPTG-inducible lac UV5 promoter. This system has been used successfully for the overproduction of many globular proteins, but in many other cases no significant overproduction can be obtained due to the toxicity of the overexpression (Studier et al., 1990; George Et al., J. Mol. Biol. 235; 424-43.
5, 1994). Also, the polymerase gene can be introduced into λ phage by infection with an integrase phage such as a commercially available CE6 phage (Novagen, Madison, USA). Other vectors include a vector containing a λ PL promoter such as PLEX (Invitrogen, NL), a vector containing a trc promoter such as pTrcHisXpressTm (Invitrogen) or pTrc99 (Pharmacia Biotech, SE), or pKK
Examples include a vector containing a tac promoter, such as 223-3 (Pharmacia Biotech) or PMAL (New England Biolabs, MA, USA).

Further, the fusion protein gene according to the present invention may comprise a secretory sequence to facilitate secretion of the polypeptide from the bacterial host, such that the polypeptide is soluble in native rather than in inclusion bodies. Produced as a peptide. The peptide may be recovered from the periplasmic space of the bacterial cell or the culture medium as appropriate.

Promoting sequences suitable for use with yeast hosts can be regulated or constitutive and are preferably derived from high-expressing yeast genes, especially the Saccharomyces cerevisiae gene. Therefore, the promoters of the TRP1, ADHI or ADHII gene, the acid phosphatase (PH05) gene, the yeast-associated pheromone gene encoding a- or α-factor, or enolase, glyceraldehyde-3
-Phosphate dehydrogenase (GAP), 3-phosphoglycerate kinase (PGK), hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase A gene encoding a glycolytic enzyme such as a triose phosphate isomerase, a phosphoglucose isomerase or a glucokinase gene promoter, S. ce
A promoter derived from the revisiae GAL4 gene, the S. pombe nmt1 gene, or a promoter derived from the TATA binding protein (TBP) gene can be used. In addition, the upstream activating sequence (UAS) of one yeast gene and the functional TAT of another yeast gene
A hybrid promoter containing a downstream promoter element such as an A box, for example, a UAS of a yeast PH05 gene and a hybrid promoter containing a downstream promoter element such as a functional TATA box of a yeast GAP gene (PH05-GAP hybrid promoter) can be used. A suitable constitutive PH05 promoter, for example, begins at nucleotide -173 and ends at nucleotide -9 of the PH05 gene
Truncated acid phosphatase PH05 promoter lacking upstream regulatory elements (UAS) such as the PH05 (-173) promoter element.

[0062] Transcription of the fusion protein gene from the vector in a mammalian host has been demonstrated by polyomavirus, adenovirus, fowlpox virus, bovine papillomavirus, avian sarcoma virus, cytomegalovirus (CMV), retrovirus and simian virus 40 (SV40). ) Is normally associated with a promoter derived from the genome of the virus, such as a promoter derived from a heterologous mammalian promoter, such as an actin promoter or a super-strong promoter, eg, a ribosomal protein promoter, and a gene encoding a component of the fusion protein. Promoters derived from such promoters can control if such promoters are compatible with the host cell system.

[0063] Transcription of the DNA encoding the fusion protein by higher eukaryotes can be increased by inserting an enhancer sequence into the vector. Enhancers are relatively direction and position independent. Many enhancer sequences from mammalian genes are known (eg, elastase and globin). However, typically,
An enhancer from a eukaryotic cell virus is used. For example, the replication origin (100-2
(70 bp) on the late side of the SV40 enhancer and the CMV early promoter enhancer. An enhancer can be spliced into the vector at the 5 'or 3' position of the coding sequence, but is preferably located 5 'to the promoter.

Conveniently, a eukaryotic expression vector encoding a fusion protein can include a locus regulatory region (LCR). LCRs have the ability to induce high levels of integration site-independent expression of a transgene integrated into host cell chromatin, particularly in vectors or transgenic animals designed for gene therapy applications. This is important when the fusion protein gene is to be expressed in a permanently transfected eukaryotic cell line in which chromosomal integration of the vector has occurred.

[0065] Expression vectors include any vector capable of expressing a nucleic acid operably linked to regulatory sequences such as a promoter region capable of expressing such DNA. Thus, an expression vector refers to a recombinant DNA or RNA construct such as a plasmid, phage, recombinant virus or other vector that results in expression of the cloned DNA upon introduction into a suitable host cell. Suitable expression vectors are well known to those of skill in the art and include vectors that are replicable in eukaryotic and / or prokaryotic cells, and vectors that are in the episomal state or that integrate into the host cell genome. For example, a DN encoding the fusion protein of the present invention
A is a vector suitable for expression of cDNA in mammalian cells, for example, pEVRF (Matthi
(1989) NAR 17, 6418).

Particularly useful in the practice of the present invention are expression vectors that cause transient expression of DNA encoding the fusion protein in mammalian cells. Transient expression usually involves the use of an expression vector that can efficiently replicate in the host cell so that the host cell accumulates multiple copies of the expression vector and then synthesizes high levels of the fusion protein. Including. For the purposes of the present invention, transient expression systems are useful, for example, to identify fusion protein variants to identify potential phosphorylation sites or to characterize functional domains of proteins. is there.

For the construction of the vector of the present invention, a usual ligation technique is used. The isolated plasmid or DNA fragment is cut, conditioned, and ligated as desired to produce the required plasmid. If desired, analysis is performed in a known manner to confirm the correct sequence in the constructed plasmid. Methods suitable for constructing expression vectors, preparing in vitro transcripts, introducing DNA into host cells, and performing assays to evaluate expression and function are known to those of skill in the art. The presence, amplification and / or expression of the gene can be determined by, for example, conventional Southern blotting, Northern blotting to quantify mRNA transcription, dot blotting (analysis of DNA or RNA), or in situ hybridization. It can be measured directly in the sample using an appropriately labeled probe based on the sequence set forth herein. Those skilled in the art will readily devise how these methods can be modified, if desired.

The present invention further provides a first nucleic acid sequence encoding a polypeptide capable of forming a coiled-coil structure, wherein the first nucleic acid sequence is linked to a promoter capable of expressing the first nucleic acid sequence in a host cell. Linked to the nucleic acid sequence,
An expression vector is provided that includes a cloning site that allows the insertion of a second nucleic acid sequence capable of expression in fusion with one nucleic acid sequence. Such vectors are useful vehicles for expressing any desired polypeptide in a fusion protein of the invention.

[0069] A further aspect of the invention provides a host cell transformed or transfected with a vector for the replication and expression of a polynucleotide of the invention. The cells are
It is selected to be compatible with the vector and can be, for example, a bacterium, yeast, insect or mammal.

[0070] Host cells, such as prokaryotic cells, yeast cells and higher eukaryotic cells, can be used for replicating DNA and producing fusion proteins. Suitable prokaryotic cells include, for example, E.co.
E. coli such as liK-12 strain, DH5a and HB101 or eubacteria such as Gram-negative or Gram-positive organisms such as Bacilli. Other suitable hosts for fusion protein-encoding vectors include filamentous fungi or yeast, such as Saccharomyces
Eukaryotic microorganisms such as cerevisiae. Higher eukaryotic cells include insect cells or vertebrate cells, especially mammalian cells. In recent years, culture (tissue culture)
Propagation of vertebrate cells in became routine. Useful mammalian host cell lines are, for example, epithelial or fibroblast cell lines such as Chinese hamster ovary (CHO) cells, NIH 3T3 cells, HeLa cells or 293T cells. The host cells referred to in this disclosure include cells in in vitro culture as well as cells in a host animal.

[0071] DNA can be stably integrated into cells, or can be transiently expressed using methods known in the art. Stably transfected mammalian cells
It can be prepared by transfecting cells with an expression vector having a selectable marker gene and growing the transfected cells under conditions that select for cells expressing the marker gene. To prepare transient transfectants, mammalian cells are transfected with a reporter gene to monitor transfection efficiency.

To produce such stable or transiently transfected cells, the cells are transfected with a fusion protein-encoding nucleic acid in an amount sufficient to produce a fusion protein. The exact amount of DNA encoding the fusion protein can be determined experimentally and can be optimized for a particular cell and assay.

A host cell is transfected, or preferably transformed, with an expression or cloning vector of the invention as described above for inducing a promoter, selecting for transformants, or amplifying a gene encoding the desired sequence. Culture in a suitable nutrient medium that has been appropriately changed. Heterologous DNA can be introduced into host cells by any method known in the art, such as calcium phosphate co-precipitation technique or transfection with a vector encoding the heterologous DNA by electroporation. Numerous transfection methods are known to those skilled in the art. Successful transfection is generally recognized when any indication about the operation of this vector occurs in the host cell. Transformation is performed using standard techniques appropriate to the particular host cell used.

Incorporation of the cloned DNA into an appropriate expression vector, a plasmid vector or combination of plasmid vectors each encoding one or more distinct genes, or transfection of eukaryotic cells with linear DNA, and transfection Sorting of transfected cells is well known in the art (eg, Sambrook et al.).
(1989) Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spr
ing Harbor Laboratory Press). The transfected or transformed cells are cultured using media and culture methods known in the art, preferably under conditions such that the fusion protein encoded by the DNA is expressed. Since the composition of suitable media is known to those of skill in the art, they can be readily prepared. Suitable media are also commercially available.

Microorganisms, and especially bacteria such as Escherichia coli, are among the most successful mediators for overexpression of both prokaryotic and eukaryotic proteins (for an overview, Hockney, 1994; Grisshammer & Tate, 1955). Please refer to). Thus, a host cell of the invention comprises a microorganism transformed with a vector encoding the fusion protein of the invention. However, membrane proteins, some cytoplasmic proteins (Dong et al., 1995) and cell division proteins (de Boer et al., 1988; Gutzman et al.
The expression systems used to express many prokaryotic proteins, including those of nucleoproteins, such as DNase, can be toxic to host bacteria in certain circumstances.

[0076] Expression of eukaryotic proteins in microorganisms can be problematic as well. Expression of such proteins can also be toxic to cells. Nevertheless, bacterial expression systems have been used in the industry, including chymosin, insulin, interferon,
It has been used to express a wide variety of proteins such as insulin-like growth factors, antibodies such as humanized antibodies or fragments thereof.

The present inventors have determined that after induction of the gene encoding the fusion protein from a culture of host cells transformed with the expression vector encoding the fusion protein of the present invention, the cells can be observed to exhibit toxic effects in the host. By harvesting and culturing such cells under sorting conditions, it is possible to recover from cultured cells capable of expressing high levels of the fusion protein without the detrimental effects normally observed in cells. I found that. Surprisingly, the effects observed are general and are observed for any target peptide encoded by the expression system. Thus, cells
In contrast to being resistant to the expression of a particular toxic gene, there is "resistance to expression system toxicity".

Thus, in a preferred aspect, the present invention provides: (a) preparing an expression system consisting essentially of a host cell transformed with an inducible expression vector encoding a fusion polypeptide of the present invention and a selectable marker. (B) culturing cells transformed with the expression system under selective pressure compatible with the selectable marker; (c) inducing the expression system to produce the fusion polypeptide such that toxic effects can be observed in the host. (D) recovering the host cells from the culture and growing them under selective pressure and induction conditions; and (e) selecting viable host cells that continue to produce the fusion polypeptide. Provide an improved method.

The generation of mutants resistant to expression system toxicity was described in 1996 in the name of the Applicant.
It is disclosed in UK Patent Application 9614700.4 filed July 12, the contents of which are incorporated herein by reference.

A fusion polypeptide comprising a coiled-coil region, and furthermore a region encoding a desired protein product, is a fusion protein of the invention. Such desired proteins include, for example, bovine oxoglutarate-malate carrier, ADP / ATP translocase, rat non-binding proteins UCP1 and UCP2, Esche
richia coli unc I, E. coli F-ATPase subunit a, bovine F-ATPase subunit epsilon and human immunodeficiency virus TAT protein.

[0081] Preferred bacterial hosts that can be used in the above methods include E.co.
li B strains or K strains such as JM109. These strains are widely available in the art from academic and / or commercial sources. The B strain lacks the lon protease, and other strains having this genotype can be used. Preferably, this strain should not be deficient in the recombinant gene.

Most preferably, this strain is BL21 (DE3) as disclosed in Studier et al. (1990).
It is. Bacteria obtained by the above-described selection method and optionally treated with a vector can be used as host cells in the present invention. Specific bacteria include E. coli
C43 (DE3) (deposited with the European Collection of Cell Cultures (ECCC) (Salisbury, Wiltshire, UK) on July 4, 1996 as B96070445); E. coli C0214 (
DE3) (June 2, 1997 in the National Collections of Industrial and Marine Bacteria)
E. coli DK8 (DE3) S (National Collections of In)
dustrial and Marine Bacteria on June 25, 1997 as NCIMB40885); or E. coli C41 (DE3) (deposited with the ECCC on July 4, 1996 as B96070444). Such bacteria, when processed, become hosts for the expression of the fusion proteins of the invention, and are particularly suitable for the expression of fusion proteins whose expression is toxic to the bacteria.

F. Selection Method Although the above selection method is fully disclosed in UK Patent Application 9614700.4, the contents of which are incorporated herein by reference, this method is preferably carried out as follows: Culturing occurs in the presence of sorting pressure, usually in the presence of an antibiotic that is metabolized by the selectable marker gene of the vector. The concentration of antibiotic used depends on the exact nature of the resistance gene and the concentration at which transformed cells are killed by the antibiotic. For ampicillin, approximately 20-200 μg per ml of culture is usually sufficient, but this can be determined empirically by one skilled in the art as needed. In general, appropriate concentrations of antibiotics can be determined using standard laboratory reference books (eg, Sambrook et al., 1989).
Can be determined by referring to FIG.

Because of the toxicity of the expression system to the cells, the host is initially required to grow cells exponentially.
Cultured under conditions that produce little or no target gene expression
Is done. For E. coli, this typically involves about 10 cells.⁶ Cells / ml, eg 10 ^Two ~Ten⁷ / Ml means growing to a density in the range of / ml. Cell density, absorbance
It can be measured using a measurement. Alternatively, the cells are allowed to grow for an appropriate amount of time, for example,
For example, it can be grown for 3 to 4 hours.

The cells may be cultured at low or high temperatures. This may be useful, for example, when the expression of the target polypeptide is linked to a temperature sensitive gene. In such a situation, the cells are first grown at a non-permissive temperature, ie, a temperature at which expression of the target gene does not occur. After culturing the cells under selective pressure, the culture is induced to express the target gene. Several inducible promoters that are functional in bacteria are available. Some promoters, such as the trpE promoter, are inducible by the presence or absence of metabolites or catabolic metabolites (ie, tryptophan in the case of the trpE promoter) in the medium. Other promoter, tac promoter or λP _R
Promoters.

[0086] However, preferred promoters are bacteriophage promoters that require a bacteriophage polymerase for expression. As noted above, a preferred promoter is the T7 promoter, which can be used for cells in which the T7 polymerase gene has been cloned and placed under the control of a separate inducible promoter. T7 polymerase is selective for its promoter binding site, thus
This is particularly useful because in the absence of T7 polymerase, little expression of the target gene occurs. The gene encoding the polymerase is introduced into cells in phage lambda such that the phage requires helper phage for integration or excision from the genome and is placed on the phage genome within the int gene. The polymerase gene is linked to a UV5 promoter inducible by isopropyl-β-D-thiogalactopyranoside (IPTG) and the production of T7 polymerase is induced by adding IPTG to the culture. Alternatively, the gene can be introduced onto λ phage by infecting an int phage such as the commercially available CE6 phage (Novagen, Madison, USA).

After induction of the gene encoding the fusion polypeptide, a toxic effect in the cells is observed, cell death begins to occur, and a suitable period of time such that the cells in culture begin to release the vector encoding the target polypeptide. Maintain the culture. Usually only 50% of cells
Cells should be maintained in liquid culture until preferably only 10%, for example 1% or 0.1%, retains the vector. This can be measured by plating two aliquots of the culture on solid media under and without selective pressure and measuring the percentage of the number of colonies that grow under selective or non-selective conditions. Things.

After growth and induction, cells in culture are harvested and grown on fresh medium under selection and induction conditions. Desirably, the fresh medium is a solid medium, typically an agar containing the nutrients necessary for cell growth. The remaining species are examined for the presence of the target gene.
We surprisingly found that some of the colonies recovered from this medium were:
It was found that the cells include cells resistant to the toxic effects of the target gene. this is,
This is in contrast to the usual practice of regarding "spent" cultures as waste products after recovery of the target polypeptide.

G. Production of the fusion protein and its processing The host cells of the invention can be cultured under conditions that allow for expression of the fusion protein.
The fusion protein can be recovered by any suitable means, for example, affinity chromatography or HPLC. When small fusion proteins are involved, HPLC is particularly suitable. A polypeptide sequence of interest can be obtained by cleaving the fusion protein using, for example, an appropriate protease, and the sequence can be recovered from a mixture of the first and second regions of the resulting fusion protein. It is. Further, in the case of a fusion protein, for example, when a coiled coil forms an aggregate,
Applications such as immunogens may be found. This avoids the need to prepare immunogenic substances by coupling small proteins and peptides to carrier proteins such as keyhole limpet hemocyanin (KLH) by separate chemical reactions.

H. Antibody Production Antisera and monoclonal antibodies can be made by using the fusion proteins of the invention directly as immunogens without additional adjuvants. According to yet another aspect of the present invention, there is provided an antibody that specifically recognizes and binds to the fusion protein of the present invention. However, more preferably, the antibody is a polypeptide that is specific for the second region of the fusion protein, ie, that is fused to the gene product of the invention to obtain its expression. Conveniently, the second region of the fusion protein is recognized by the antibody when in its native state. Thus, if the second region is an isolated peptide or domain from a large protein, that peptide or domain will be recognized by the antibodies of the invention for the entire large protein.

The present invention further provides a) a step of immunizing an animal with the fusion protein according to any one of claims 1 to 7; and Provided is a method for preparing an immunoglobulin, comprising a step of recovering the immunoglobulin.

Antibodies (or immunoglobulins) can be isolated in crude preparation, ie, antiserum, by affinity chromatography on the fusion protein or a protein from which a region of the fusion protein is derived. Conveniently, this area is a second area. Also, if the antibody recognizes both a coiled coil and a fusion protein, the antibody can be isolated using a separate fusion between the fusion protein and a separate coiled coil structure. The animal used for antibody production can be any animal commonly used for such purposes, especially mammals. In particular, mice, rats, guinea pigs and rabbits are mentioned.

In the following description, both an antibody specific to a fusion protein and an antibody raised against a fusion protein specific to one region of the fusion protein are meant to be “anti-fusion protein” antibodies. It is.

The antibodies of the present invention may be whole class whole antibodies, such as IgE and IgM antibodies, but IgG antibodies are preferred. In addition, the invention includes antibody fragments such as Fab, F (ab ') 2, Fv and ScFv. Small fragments, such as Fv and ScFv, have properties that are favorable for diagnostic and therapeutic applications due to their small size and resulting excellent tissue distribution.

The antibodies of the present invention are particularly desirable for diagnostic and therapeutic uses. Thus, they can be modified antibodies containing effector proteins such as toxins or labels. Particularly preferred are labels that allow imaging of antibody distribution in tumors in vivo. Such a label can be a radioactive or radiopaque label, such as a metal particle that is easily visualized within a patient. Further, it may be a fluorescent label or other label that can be visualized in a tissue sample collected from a patient.

The antibodies of the present invention can be improved by using recombinant DNA technology. Thus, chimeric antibodies can be constructed to reduce their immunogenicity in diagnostic or therapeutic applications. In addition, immunogenicity was determined by CDR transplantation (European patent application 0 2
39 400 (Winter)) and optionally humanization of the antibody by framework modification. Also, human antibodies can be synthesized using phage display selection techniques.

The antibodies of the present invention can be obtained from animal serum or, in the case of a monoclonal antibody or a fragment thereof, produced in cell culture. By using recombinant DNA technology, antibodies can be produced in bacterial or preferably mammalian cell culture according to established procedures. Preferably, the selected cell culture system secretes the antibody product.

Accordingly, the present invention relates to an expression cassette comprising a promoter operably linked to a first DNA sequence encoding a signal peptide, linked in proper reading frame to a second DNA sequence encoding an antibody of the invention. Culturing a host, such as E. coli or a mammalian cell, transformed with a hybrid vector containing
And a method for producing the antibody of the present invention, comprising isolating the protein.

In vitro growth of hybridoma cells or mammalian host cells is performed in a suitable medium. Here, the medium is a normal standard medium, for example, optionally, mammalian serum, such as fetal bovine serum, or trace elements and growth maintenance supplements, such as feeder cells such as normal mouse peritoneal exudate cells, spleen cells, Bone marrow macrophages, 2-
Dulbecco's modified Eagle's medium (DMEM) or RPMI 1640 medium supplemented with aminoethanol, insulin, transferrin, low-density lipoprotein, and oleic acid. Growth of a host cell, which is a bacterial or yeast cell, is also carried out in a suitable medium known in the art, for example, for bacteria, medium LB, NZCYM, NZYM, NZM, Te.
rrific Broth, SOB, SOC, 2 × YT, or M9 minimal medium, Yeast for yeast
Performed in YEPD, minimal medium, or complete minimal dropout medium.

In vitro production results in relatively pure antibody preparations, which can be scaled up to obtain large quantities of the desired antibody. Techniques for bacterial cell, yeast or mammalian cell culture are known in the art, for example, homogeneous suspension culture in an airlift reactor or continuously stirred reactor, or in hollow fibers, microcapsules, agarose microbeads or ceramic cartridges Immobilized or captured cell culture above.

[0101] Large quantities of the desired antibodies can also be obtained by growing mammalian cells in vivo. To this end, hybridoma cells producing the desired antibody are injected into a histocompatible mammal to cause growth of the antibody-producing tumor. Prior to injection, the animals are optionally primed with a carbohydrate, especially a mineral oil such as pristane (tetramethyl-pentadecane). After one to three weeks, the antibodies are isolated from the body fluids of those mammals. For example, hybridoma cells obtained by fusion of appropriate myeloma cells with antibody-producing spleen cells derived from Balb / c mice, or transfected cells derived from the hybridoma cell line Sp2 / 0 that produce the desired antibody, are optionally primastane. Is injected intraperitoneally into Balb / c mice pre-treated with, and ascites fluid is collected from the animals after 1-2 weeks.

The cell culture supernatant is screened for the desired antibody, preferably by immunofluorescent staining of cells expressing the fusion protein, by immunoblotting, by an enzyme immunoassay, such as a sandwich or dot assay, or by a radioimmunoassay. I do.

For the isolation of antibodies, immunoglobulins in culture supernatants or ascites fluid are for example:
It can be concentrated by precipitation with ammonium sulfate, dialysis against a hygroscopic material such as polyethylene glycol, filtration through a selective membrane and the like. If necessary and / or desired, the antibodies can be purified by conventional chromatographic methods, such as gel filtration, ion exchange chromatography, chromatography on DEAE-cellulose and / or immunoaffinity chromatography, such as the relevant fusion protein (the relevant part of the fusion protein). Alternatively, it is purified by affinity chromatography using protein-A. The present invention further relates to a hybridoma cell secreting the monoclonal antibody of the present invention. Preferred hybridoma cells of the present invention are genetically stable,
The monoclonal antibodies of the present invention of the desired specificity can be secreted and activated from cryofreeze cultures by thawing and recloning.

The present invention provides a method for immunizing a suitable mammal, for example, a Balb / c mouse, with a purified fusion protein, or a cell having the fusion protein, and immunizing the antibody-producing cells of the immunized animal with cells of a suitable myeloma cell line. And a method for preparing a hybridoma cell line secreting a monoclonal antibody specific to the fusion protein, characterized by cloning the hybrid cell obtained by the fusion. For example, spleen cells of a Balb / c mouse immunized with the fusion protein are fused with cells of the myeloma cell line PAI or myeloma cell line Sp2 / 0-Ag14, and the resulting hybrid cells are secreted for secretion of a desired antibody. And positively hybridoma cells are cloned.

Balb / c mice are immunized by subcutaneous and / or intraperitoneal injection of an appropriate amount of the fusion protein of the present invention several times, eg, 4-6 times, for several months, eg, 2-4 months. A hybridoma characterized in that spleen cells are taken from the immunized mouse 2-4 days after the last injection and fused with cells of the myeloma cell line PAI in the presence of a fusion promoter, preferably polyethylene glycol. Cell line preparation methods are preferred. Preferably, the myeloma cells are transformed to about 30 with a molecular weight of about 4000.
It is fused with a 3-20 fold excess of spleen cells from immunized mice in a solution containing ~ 50% polyethylene glycol. After fusion, the cells were grown in a suitable medium, such as those described above, periodically supplemented with a selection medium, eg, HAT medium, to prevent normal myeloma cells from overgrowing the desired hybridoma cells.

The present invention also relates to a recombinant nucleic acid comprising an insert encoding the H chain variable domain and / or the L chain variable domain of an antibody specific for the fusion protein of the present invention. By definition, such DNAs include single-stranded coding DNA, double-stranded DNA consisting of the coding DNA and its complementary DNA, or their complementary (single-stranded) DNA.

Furthermore, the DNA encoding the H chain variable domain and / or the L chain variable domain of the antibody specific to the fusion protein may be a genuine DNA sequence encoding the H chain variable domain and / or the L chain variable domain, or It may be enzymatically or chemically synthesized DNA having the mutant. Mutants of authentic DNA are DNAs encoding the H chain variable domains and / or L chain variable domains of the above antibodies wherein one or more amino acids have been deleted or replaced with one or more other amino acids. It is. Preferably, the modification is outside the CDRs of the heavy and / or light chain variable domains of the antibody. Such mutant DNAs are also contemplated to be silent mutants, in which one or more nucleotides are replaced with other nucleotides having new codons encoding the same amino acid. Such a mutant sequence is also a degenerate sequence. Degenerate sequences are degenerate within the context of the genetic code in which an unlimited number of nucleotides have been replaced with other nucleotides so that no change in the originally encoded amino acid sequence occurs. Such degenerate sequences may be selected from particular restriction sites and / or specific codons that a particular host, particularly E. coli, may prefer to obtain optimal expression of the heavy and / or light chain mouse variable domains. May be more useful.

[0108] The term mutant is intended to include DNA mutants obtained by in vitro mutagenesis of authentic DNA by methods known in the art. For assembly of a complete tetrameric immunoglobulin molecule and expression of a chimeric antibody, the recombinant DNA insert encoding the heavy and light chain variable domains is fused to the corresponding DNA encoding the heavy and light chain constant domains, It is then transferred into a suitable host cell, for example after integration into a hybrid vector.

Accordingly, the present invention relates to human gamma constant domains, such as γ1, γ2, γ3 or γ4
Of an antibody specific to the fusion protein of the present invention, preferably fused to γ1 or γ4
It also relates to a recombinant DNA comprising an insert encoding a heavy chain mouse variable domain. Similarly, the present invention relates to a recombinant D comprising an insert encoding the light chain mouse variable domain of an antibody specific for a fusion protein fused to a human constant domain κ or λ, preferably κ.
About NA.

[0110] In another embodiment, the present invention provides that the heavy and light chain variable domains are:
A signal sequence that facilitates antibody processing in host cells and / or a DNA that encodes a peptide that facilitates antibody purification and / or a DNA that encodes a cleavage site and / or a DNA that encodes a peptide spacer and / or encodes an effector molecule And a recombinant DNA encoding the recombinant DNA, optionally linked by a DNA insert encoding a spacer group.

[0111] DNA encoding effector molecules is contemplated to be DNA encoding effector molecules useful in diagnostic or therapeutic applications. Accordingly, effector molecules that are toxins or enzymes, particularly enzymes that have the ability to catalyze the activation of prodrugs, are particularly desirable. The DNA encoding such an effector molecule has the sequence of the DNA encoding the native enzyme or toxin or a mutant thereof, and can be prepared by methods well known in the art.

The antibodies and antibody fragments of the present invention are diagnostically and therapeutically useful. Accordingly, the present invention provides a therapeutic or diagnostic composition comprising the antibody of the present invention. In the case of a diagnostic composition, the antibody is preferably provided with a means for detecting the antibody, which may be enzymatic, fluorescent, radioisotope or other means. The antibody and the detection means are used simultaneously in a diagnostic kit for diagnosis.
It may be provided for simultaneous separate or continuous use.

I. Use in NMR studies The fusion proteins of the invention have a minimal fusion partner. One advantage is that
The fusion protein can be used directly in NMR experiments without the resulting spectrum being interfered by the fusion partner. NMR analysis is described, for example, in K. Wurtrich, "NMR of Protein and Nucleic Acids", Wiley
, New York, 1986, which is incorporated herein by reference, can be performed according to techniques and methods known in the art. The invention will now be described with reference to the following examples.

General expression of over- engineered proteins and bacterial cell disruption One newly transformed colony of the expression host E. coli C41 (DE3) was
Inoculate Y medium. When the absorbance of the culture at 600 nm reaches 0.6, expression of the fusion protein is induced by adding IPTG (0.7 mM final concentration). After the induction, the growth temperature was lowered to 25 ° C, and the culture was performed for 18 hours. Cells are then harvested by centrifugation, resuspended in TEP buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA, and 0.001% phenylmethylsulfonyl fluoride) and passed twice through a French pressure cell. Destroy by. Centrifuge the disrupted cells (60 min, 100,000 × g).

Purification of soluble fusion protein on S-Sepharose If the overexpressed protein is soluble in the bacterial cytosol, a column of S-Sepharose equilibrated in TEP buffer with centrifuged supernatant of disrupted bacterial cells Add to The protein bound to the column is eluted with a linear gradient of sodium chloride (0-1M). Most of the fusion proteins elute at about 0.9 M sodium chloride
-Elute from the column at a salt concentration of about 0.5 M sodium chloride, except for ADPI. Check protein purity by SDS-PAGE. If impurities are present, the appropriate fraction containing the fusion protein is loaded onto a column of Ni-NTA resin (supplied by Quiagen, Chatworth, CA 91111, USA; NTA is nitrilotri-triacetic acid).

Purification of fusion protein forming inclusion bodies If-ε. The pellet obtained by centrifugation of the disrupted cells of E. coli C41 (DE3) was
Redissolve in PBS containing guanidine hydrochloride and 0.33 M sodium chloride. Remove insoluble material by centrifugation (40,000 g, 10 minutes), and purify the fusion protein from the supernatant by reverse-phase HPLC or Ni-NTA column chromatography as described below. (a) Reversed phase HPLC. The solution of If-ε was equilibrated in 0.1% aqueous trifluoroacetic acid
Apply to a column of Aquapore RP-300 (Applied Biosystems; particle size 7 microns, pore size 330Å; inner diameter 10 cm x 2.1 mm). The column is eluted with a linear gradient of acetonitrile in 0.1% aqueous trifluoroacetic acid (flow rate 0.1 ml / ml). Monitor the absorbance of the eluate from the column at 225 nm. Protein was eluted from the column with 41% acetonitrile. (b) Ni-NTA column chromatography. A sample of If-ε dissolved in 6M-guanidine hydrochloride is added to a column of nNi-NTA resin (bed volume 2 ml). Equilibrate the column in PBS containing 0.33 M sodium chloride. The column was first washed with PBS containing 5 mM imidazole (20 ml
) And then with PBS containing 50 mM imidazole (20 ml)
Elute If-ε. The protein in the eluate is detected by monitoring the absorbance at 225 nm. Confirm protein purity by SDS-PAGE. If-UCP270. This protein (containing a histidine ₆ tag at its C-terminus) is
It is expressed in 41 (DE3) and also forms inclusion bodies in bacterial cytoplasm. The culture is harvested by centrifugation and the cells are resuspended in PBS buffer containing 0.3 M sodium chloride. Pass the cells through a French press twice and centrifuge the non-destructive cells at low speed (2,000 xg, 1
0 min). The inclusion bodies are recovered by centrifugation (10,000 g, 10 minutes). Resuspend the inclusion body pellet in PBS buffer until the protein concentration is 2 mg / ml. 4m
of the suspension was centrifuged (10 min, 10,000 × g) and the resulting inclusion body pellet was buffered with buffer A (containing 0.1 M sodium phosphate, 0.01 M Tris-HCl, 8.0 and 6 M guanidine hydrochloride). Solubilized. Remove any remaining insoluble material by centrifugation (10,000 xg, 10 minutes). The supernatant is mixed with 2 ml of a slurry of Ni-NTA resin equilibrated in the same buffer. The slurry is stirred at room temperature for 45 minutes and then poured onto a 5 ml chromatography column. The column is washed successively with 10 volumes of buffer A, pH 8.0, and 5 volumes of buffer B (8 M urea, 0.1 M sodium phosphate, 0.01 M Tris-HCl, pH 6.8). The fusion protein was treated with 8M urea, 0.1M sodium phosphate, 0.01M Tris, pH4
Elute by washing the column with 4 ml of buffer C containing .5. The surfactant lauryl-dimethylamine oxide (LDAO; final concentration 1%) was added to the eluted fractions, and the following buffer: buffer A containing 0.15% LDAO and 4M urea, pH 8.0 The second is a buffer A containing 0.15% LDAO and 0.5 M urea, pH 8.0; the third is a buffer A containing 0.15% LDAO, pH 8.0, and three successive dialysis steps (each 3 Time) to remove urea.

Purification of fusion protein on Ni-NTA column after S-Sepharose chromatography Fractions containing fusion protein were pooled and Ni-NTA resin pre-equilibrated with PBS buffer containing 0.3 M sodium chloride Incubate with 2 ml at 4 ° C for 45 minutes. The mixture is then poured onto a 10 ml chromatography column, and P containing 5 mM imidazole is added.
Wash with BS buffer (20 ml). Additional fusion protein without C-terminal His tag
Elute with a buffer containing 50 mM imidazole and elute the fusion protein with an additional C-terminal His tag with a buffer containing 500 mM imidazole. The fusion protein is dialyzed against PBS buffer and concentrated by ultrafiltration over Amicon column 3.

[0118] Example 1 Construction pMW T7 expression vector coiled-coil fusion expression vectors (Studier et al., 1990; Way et al., 1990), and the IF ₁ amino acid 44 to
It was adjusted to express a fusion protein containing a coiled coil fusion partner consisting of 84. By this end, the amino acid sequence shown in SEQ ID NO: 1 (starting ATG (Me
consisting of amino acids 44-84 of IF ₁ to t) preceded) is connected downstream of the T7 promoter in pMW by ligating the sequence to Nde1 restriction site. Inserting a cleavable linker GSPKL downstream of IF ₁ sequence. This provides a BamH1 and HindIII restriction endonuclease site. This sequence is part of the reverse primer used for vector construction and corresponds to the BamHI and HindIII sites in the vector.

Another vector is constructed in the same manner, but incorporating another cleavable linker: GSIEGR (cleavable by factor Xa) and GSSLVPRGSGS (cleavable by thrombin). FIG. 1, lane 1, shows the expression of only the fusion partner + cleavable linker. The expression vector is transformed into E. coli C41 (DE3) cells (UK patent application 9614700.4; Miro
ux and Walker, 1996). One transformant colony was inoculated into 500 ml 2 × TY medium in the presence of ampicillin (100 μg / ml) and the culture had an OD ₆₀₀ of 0.5.
Grow at 37 ° C. to 6 At this stage, fusion partner expression is induced by adding 0.7 mM IPTG to the culture. Cells were harvested 16 hours after induction and SDS-PA
Dissolve in GE solubilization buffer. Mix 7.5 μl of the culture with an equal volume of SDS-PAGE buffer and load on a 12-22% SDS-PAGE gel without further purification. The gel is then stained with Coomassie Blue 83 dye.

The expressed polypeptide is visible as a major component of all cellular proteins in the bacterium. General utility vector The above vectors are further modified by the addition of MCS and His tags to obtain expression vectors that can be used to routinely clone and express various peptides. His tag is one that facilitates the isolation of the fusion protein on a nickel column, not strictly necessary since the IF ₁ forty-four to eighty-four sequence itself contains five His residues.

Example 2 Expression of Fusion Protein Amino acids 1-39 and 118-15 of subunit a of E. coli F ₁ F ₀ ATP synthase
5 and E. coli F ₁ F ₀ amino acids of subunit I of ATP synthase 1-13, and
A nucleic acid sequence encoding the 55-75, 44 of IF ₁ in the expression vector described in Example 1
The fusion polypeptide is expressed by cloning directly onto the ~ 84 sequence GSPKL extension. The results of the experiment are shown in lanes 2 to 5 in FIG.

The expression vector is transformed into E. coli C41 (DE3). One of the transformant colonies was inoculated in the presence of ampicillin (100 [mu] g / ml) in 500 ml 2 × TY medium, OD _{6 00} is grown at 37 ° C. until 0.6 cultures. At this stage, fusion partner expression is induced by adding 0.7 mM IPTG to the culture. Cells are cultured at a low temperature of 25 ° C. and harvested by centrifugation 18 hours after induction, or 3 hours after induction in the case of IF ₁ (44-84) -a (1-39) fusion. 7.5 μl of culture for 12-22 without further purification
Load on% SDS-PAGE gel. The gel is then stained with Coomassie Blue 83 dye.
The expressed polypeptide is visible as a major component of all cellular proteins in the bacterium.

Table 1 shows the results of expression experiments performed on several fusion proteins. In two cases, the His tag was used to further facilitate isolation on a nickel column as it contained a fusion with a portion of the rat non-mitochondrial-bound protein. Cow F ₁ F ₀ A
Two fusions with the epsilon subunit of TP synthase were made using alternative cleavable sequences, namely those for factor Xa and thrombin.

In all cases, E. coli C41 (DE3) cells were transformed as described above and the protein was isolated from the cytosol in a soluble state or as inclusion bodies as indicated. The inclusion bodies are solubilized in either 6M-guanidinium chloride or 8M-urea and, after solubilization, centrifuged to remove membranes and insoluble materials. Reversed-phase HPLC column (C8 Aquapore) in which supernatant containing solubilized protein was equilibrated in 0.1% trifluoroacetic acid.
RP-300, 7 μm particles, particle size 300 mm; inner diameter 10 cm × 2.1 mm), and eluted with a linear gradient of acetonitrile. Eluted peptides are detected by monitoring the absorbance of the eluate at 220 nm and analyzing them by SDS-PAGE.

In each case, the mass of the fusion protein was calculated in a theoretical manner and the Perkin-Elmer
Measured experimentally by electrospray ionization mass spectrometry using a Sciex API III + triple quadrupole mass spectrometer. A purified protein or peptide sample is dissolved in water or 0.1% trifluoroacetic acid, introduced into the inlet stream and sprayed into the device. This stream consisted of a 50% aqueous solution of acetonitrile. Spectrum from mass range 10 to
2,400 (Molecular Biology, Vol. 61: "Protain and Peptide Analysi
s by Mass Spectrometry ": Edited by JRChapman, Humana Press, 1996; see M. Mann and M. Wilm (1995) Trends Biochem Sci. 20, 219-224). This indicates that the polypeptide was not destroyed at all during the synthesis procedure.

[0126]

[Table 1]

[0127] Italicized letters indicate extra amino acids, bold letters indicate the name of the protein, and bold numbers in parentheses indicate the position of the peptide in the protein. It was used The following abbreviations: Inhibitor: F ₁ F ₀ -ATP bovine inhibitor of _{synthase; Eca: E.coli F 1 F 0} -ATP subunit synthase _{a; uncl: E.coli F 1 F} 0 -ATP sheet synthase Subunit I; OGCP: bovine mitochondrial oxoglutarate carrier; AA
C: Usiadenine nucleotide translocator; UCPI: Rat mitochondria non-binding protein 1; UCP2: Rat mitochondria non-binding protein 2; ADPI: HIV
-TAT protein derived peptides; epsilon: subunit of bovine F ₁ F ₀ -ATP synthase; e: subunit of bovine F ₁ F ₀ -ATP synthase; IB: inclusion bodies; m: membrane protein; g: Spherical Protein; aa: amino acid; LDAO: lauryl dimethylamine oxide.

[0128] was ligated to a nucleic acid cDNA fragment encoding some randomly chosen peptide expression of the fusion peptide into an expression vector containing the IF ₁ fusions. The expression of these vectors is shown in FIG. 2; the procedure was identical to the experiment reported in FIG. The expressed peptides are shown in Table 2.

[0129]

[Table 2]

Example 3 Purification of Fusion Protein Nickel Column FIG. 5 shows the purification of the fusion protein of the present invention by a nickel column and reverse phase HPLC. 1 / indicates purification of protein from inclusion bodies: If-a1 occurs as inclusion bodies, is solubilized using 6M guanidinium chloride, and purified by reverse phase HPLC as in FIG. The If-UCP270 fusion protein is solubilized in the presence of urea and purified on a nickel column in the presence of an LDAO detergent (see General Operation for details).

FIG. 2 / shows the purification of the three fusion proteins If-UCP1P1, If-UCP2P2 and If-UCP3P3 on a nickel column. These fusions are soluble in bacterial cytoplasm and purified by anion exchange chromatography on S-Sepharose ("General procedure").
) And then purified by affinity chromatography on a nickel column. All protein samples are analyzed by SDS-PAGE after purification. The gel is stained with Coomassie blue dye 83.

Thus, nickel columns provide a convenient and efficient means of purifying the fusion proteins of the present invention. Reverse Phase HPLC FIGS. 3 and 4 illustrate the purification of the fusion proteins of the invention by reverse phase HPLC ("General Procedure"; see also Table 1).

Polypeptides isolated by reverse phase HPLC are isolated from bacterial host cells in the form of inclusion bodies and about 150 μg of substance dissolved in 0.1 M Tris HCl, pH 8.0, 6 M guanidinium chloride. The sample was injected onto an Aquapore RP300 column using an HP 1090 liquid chromatograph. 2 shows the purification of If-I1, If-I2, If-a1 and If-A3 (see also Table 1).

[0134] The fusion proteins containing Example 4 Antibody Preparation E.coli F ₁ F ₀ -ATP amino 44-84 of IF ₁ fused via a linker GSPKL residues 1 to 39 of a subunit of synthase , Anti-F ₁ F ₀ -A
Used directly for rabbit challenge to produce TP synthase antibodies. Immunization is performed according to established techniques ("Antibodies. A Laboratory Ma
(See, "Nual", E. Harlow and D. Lane (1988) Cold Spring Harbor, USA). Rabbits were injected with purified fusion protein (about 1 mg) in the presence of complete Freund's adjuvant. Booster injections in addition to complete Freund's adjuvant were given 4 weeks after the first injection Antibodies were isolated from rabbit sera and tested for reactivity with the a subunit of F ₁ F ₀ -ATP synthase. By this method, an antibody having the ability to selectively bind to the selected polypeptide is obtained.

Example 5 Direct Application of Fusion Proteins to NMR The fusion proteins are subjected to NMR analysis according to conventional techniques, except that the fusion partner is not separated from the polypeptide to be analyzed (K. Wurtrich, "NMR of Pro
Teins and Nucleic Acids ", Wiley, New York, 1986. Proton and ¹³ C NMR analysis confirm the structure of the desired protein. Relevant spectra have at most minimal spectral peaks due to the fusion partner.

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109.

[Sequence list]

SEQ ID NO: 1 Sequence length: 141 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: cDNA to mRNA Hypothetical: NO Antisense: NO Fragment type: C-terminal fragment Origin Organism name: Bos bovis Direct origin Clone name: IF1 ATPase inhibitor Sequence features Symbol indicating the feature: CDS Location: 1..141 Sequence SEQ ID NO: 2 Sequence length: 47 Sequence type: Amino acid Topology: Linear sequence

[Procedural Amendment] Submission of translation of Article 34 Amendment of the Patent Cooperation Treaty

[Submission date] January 7, 2000 (2000.1.7)

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] Claims (full text)

[Correction method] Change

[Correction contents]

[Claims (full text)]

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12N 1/19 C12N 1/21 1/21 G01R 33/24 5/10 (C12N 1/21 G01R 33/24 C12R 1:19) // (C12N 1/21 C12N 15/00 A C12R 1:19) 5/00 A (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR) , GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD) , TG), AP (GH, GM, KE, LS, MW, SD, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AM , AT, AU AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GE, GH, GM, HU, ID, IL, IS, JP , KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, ZW (72) Inventor Miroux, Bruno UK CB2 2QH Cambridge , Hills Road, Medical Research Council Center, MRC Laboratory of Molecular Biology

Claims (20)

[Claims] 1. A fusion protein comprising: a) a first region capable of forming a coiled coil structure; and b) a second region containing a polypeptide sequence of interest and not naturally related to the first region. 2. The fusion protein according to claim 1, further comprising a cleavable linker region between the first region and the second region. 3. The fusion protein according to claim 1, wherein the first region is at or near the N-terminus of the protein. 4. The fusion protein according to claim 1, wherein the length of the polypeptide sequence of interest is 2 to 100 amino acids. 5. The fusion protein according to claim 1, wherein the polypeptide sequence of interest is a mammalian hormone. 6. The fusion protein according to claim 1, wherein the first region comprises 4 to 10 7-residue repeating units capable of forming a coiled-coil structure. 7. The fusion protein according to claim ₁ , wherein the first region comprises all or a part of a bovine IF ₁ ATPase inhibitor protein. A nucleic acid encoding the fusion protein according to any one of claims 1 to 7. 9. An expression vector comprising the nucleic acid of claim 8 operably linked to a promoter. 10. A first nucleic acid sequence encoding a polypeptide capable of forming a coiled coil structure operably linked to a promoter capable of expressing the first nucleic acid sequence in a host cell; and a nucleic acid sequence linked to the nucleic acid sequence. An expression vector comprising a cloning site into which a second nucleic acid sequence can be inserted to have the ability to be expressed in a fusion with the first nucleic acid sequence. 11. A host cell transformed with the expression vector according to claim 9 or 10. 12. (i) transforming the host cell of claim 11, wherein the method comprises culturing the host cell under conditions that cause expression of the fusion protein from the expression vector in the host cell. And (ii) recovering the fusion protein. 13. The method according to claim 12, wherein the host cell is E. coli. 14. The method of claim 13, wherein the expression vector comprises a bacteriophage T7 promoter. 15. The fusion protein further comprises a protease cleavable linker region between the first region and the second region, the method comprising cleaving the protein with the protease cleavable linker to recover the second region. The method according to any one of claims 12 to 14, further comprising: A polypeptide prepared by the method according to any one of claims 12 to 15. 17. Use of a polypeptide capable of forming a coiled-coil structure as a fusion partner in the construction of a fusion protein. 18. Use according to claim 17, wherein the fusion protein is a fusion protein according to any one of claims 1 to 7. 19. Use of the fusion protein according to any one of claims 1 to 7 in NMR studies. 20) a) immunizing an animal with the fusion protein according to any one of claims 1 to 7, and b) recovering an immunoglobulin specific to the region of the fusion protein from the serum of the animal. A method for preparing an immunoglobulin, comprising the step of collecting.