JP2005065564A - Sensor protein - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a general method with which a specific bond region having heterogeneity is transferred to protein having specific catalytic activity in higher degree of freedom so that the activity of the protein having catalytic activity is specifically modulated and to obtain a highly specific sensor protein obtained by the method. <P>SOLUTION: A natural type protein having catalytic activity is varied by circular permutation by a genetic engineering technique. Namely, linkers link the N and C terminals of the natural type protein by using a genetic engineering technique and then a variant to which new N and C terminals are transferred to the neighborhood of the active site is prepared. A specific bond region of another protein is added to either or both of the new N and C terminals of the variant. Consequently, the specific bond region derived from any of various heterologous proteins is extremely readily transferred to the fixed position of the active site periphery of the sensor protein to prepare hybrid protein. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a sensor protein imparted with a target specificity. The present invention also relates to the use of the sensor protein in a homogeneous assay. Furthermore, the present invention relates to a kit for homogeneous assay containing the sensor protein.

  Proteins having a specific catalytic activity typified by enzymes, in particular, proteins that produce easily detectable products have been widely used in the measurement field using biotechnology. As a typical example of such a measurement, so-called “homogeneous immunoassay” is also widely used, and the homogeneous immunoassay is extremely accurate and sensitive based on the high specificity of the antibody and the high detection sensitivity of the enzyme. Inspection is realized. That is, in a homogeneous immunoassay, the enzyme activity is modified to be modulated by reaction with a particular antibody. For example, a hapten of another substance is chemically bound on an enzyme, and the activity of the enzyme is modulated by the binding of an antibody against the hapten and the hapten on the enzyme. Through the activity modulation, the presence of the test substance in the sample is suitably identified or quantified based on a competitive immunoassay protocol.

  As examples of such enzymes, alkaline phosphatase, horseradish peroxidase, β-galactosidase, glucose 6-phosphate dehydrogenase (G6PDH) and the like are known. In particular, alkaline phosphatase is a low-specificity phosphomonoesterase and is frequently used as a marker for hepatobiliary and bone diseases in clinical examinations, or as an enzyme label for immunoassay, in addition to the homogeneous immunoassay described above. E. coli enzyme PhoA, which is a representative example of the alkaline phosphatase, is a dimer enzyme that is strong against heat and protease, and is known to maintain stability and activity even when an epitope peptide is inserted into its loop part in previous studies. ing. By inserting an epitope that interacts with another molecule (for example, an antibody) into such a loop portion existing in the vicinity of the active site, the active site is sterically blocked or changed in the presence of the interacting molecule. It has been found that a system for controlling enzyme activity can be produced.

  Specifically, in Non-Patent Document 1, by inserting an epitope (13 amino acid residues) of HIV-1 gp120 protein between amino acids 407 and 408 of PhoA, the inserted protein is anti-gp120. It is described that the activity of the enzyme was modulated due to the reactivity with the monoclonal antibody and the binding with the antibody. That is, the PhoA enzyme having the gp120 epitope insertion as described above maintains the phosphatase activity equivalent to that of the wild type in the absence of the anti-gp120 monoclonal antibody. Has been found to decrease by 40 to 50%. Such a modulation of activity is caused by a catalyst based on the interference of the enzyme substrate due to steric hindrance to the active site of the modified PhoA enzyme due to the binding of the monoclonal antibody, or the fluctuation of the three-dimensional structure of the enzyme itself due to the binding of the antibody. The latter is presumed to be caused by a change in the rate constant, and the latter is considered to be extremely dominant.

In particular, in the other two inserts described in this document, the gp120 epitope (13 or 15 amino acid residues) is substituted between amino acid residues 91 and 93 of PhoA. The insert showed activity similar to that of the natural enzyme, but unlike the insertion between amino acids 407 and 408, the activity was not modulated by the binding of the anti-gp120 monoclonal antibody. Yes. The substitutional insertion of PhoA at amino acid residues 91-93 is similar to the insertion between amino acid residues 407-408, and the gp120 epitope is located around the active site of the enzyme. Thus, the insertion of an epitope that causes steric hindrance around the active site may not change the activity due to the binding of the antibody. Furthermore, instead of the insertion between the amino acid residues 407-408, the insertion site is changed to the C-terminal side The authors of Non-Patent Document 1 indicate that the main cause of the activity modulation is not based on steric hindrance but rather the enzyme. It is concluded that it may be due to the three-dimensional structure change.
Catherine Brennan et al., Protein Engineering, Vol. 7, no. 4, pp. 509-514 (1994)

  Therefore, the present invention provides a general technique for introducing a heterogeneous specific binding region onto a specific protein having catalytic activity with a higher degree of freedom, thereby specifically modulating the activity of the protein having catalytic activity. An object of the present invention is to provide a highly specific sensor protein obtained by the method. In this modulation, not only a change in the catalytic activity itself depending on the presence of the specific binding region-partner binding, but also a steric hindrance effect on the proximity of the substrate to the catalytic active site by the binding of the specific binding region-partner can be suitably imparted.

  In the general method for producing the sensor protein of the present invention, first, a natural type protein having catalytic activity is modified by genetic engineering by circular permutation (Circular Permutation). That is, using a genetic engineering technique, a N / C terminus of a natural protein is connected by a linker, and then a mutant in which a new N / C terminus is introduced near the active site is prepared. Specific binding regions of other proteins are added to either or both of the new N / C termini of the mutant. As a result, hybrid proteins can be prepared very easily by introducing specific binding regions derived from various heterologous proteins at desired locations around the active site of the sensor protein.

  The catalytic activity of the sensor protein thus obtained is modulated based on the interaction between the specific binding region derived from the heterologous protein introduced therein and the specific binding partner for the region. Based on this modulation, a very specific and sensitive biochemical assay is achieved.

  That is, in the first aspect of the present invention, the natural N-terminal and C-terminal of a natural protein having a detectable catalytic activity are linked by a linker, and new N-terminal and C-terminal are formed around the catalytic active site of the protein. And a sensor protein produced by binding a heterologous specific binding region to the new N-terminus and / or C-terminus. The protein having a detectable catalytic activity is preferably an enzyme, and alkaline phosphatase is particularly preferable. For example, when the heterogeneous specific binding region is an epitope of another protein, the sensor protein can be suitably applied to a homogeneous competitive immunoassay protocol using an antibody against the epitope.

Accordingly, in another aspect of the invention, a homogeneous assay method for identifying and / or quantifying the presence of a test substance in a sample, the assay method comprising:
(1) The original N-terminal and C-terminal of a natural protein having a detectable catalytic activity are linked by a linker to generate new N-terminal and C-terminal around the catalytic active site of the protein, and the new protein Using a sensor protein produced by binding a heterogeneous specific binding region derived from a test substance to the N-terminus and / or C-terminus,
(2) contacting a sample presumed to contain a test substance with the sensor protein in the presence of at least one specific binding partner for the heterogeneous specific binding region; and (3) a sensor by the contact Measuring the modulation of the catalytic activity of the protein,
The homogeneous assay method is provided.

  Specific binding partners can include antibodies and receptors. That is, when the heterogeneous specific binding region on the sensor protein is an epitope of the heterologous protein, the partner is an antibody against the epitope, and when the heterogeneous specific binding region is a peptide hormone or the like, The partner may be a receptor for the hormone.

  The present invention also provides a kit comprising the sensor protein of the present invention and a specific binding partner useful for the assay.

  Finally, the present invention provides a circular permutation of E. coli alkaline phosphatase, a novel protein, as a suitable example of the circular permutation protein important in producing the sensor protein of the present invention. As an example of the sensor protein, a fusion protein of the circular permutant and the osteocalcin epitope is provided.

The term “detectable catalytic activity” as used herein refers to any catalytic activity exhibited by a particular protein, preferably an enzyme. That is, the catalytic activity refers to an activity in which a protein chemically or physically converts a specific substrate to produce a product having a structure or physicochemical property different from that of the substrate. Thus, the activity can change the conformation and topology of the substrate as well as changes in the chemical structure of the substrate. Changes in the chemical structure, conformation and / or topology imparted to the product by the catalytic activity are detected as direct or indirect signals based on various physicochemical properties of the product. obtain. Furthermore, a specific protein may require a coenzyme for exerting the catalytic activity. The change in the coenzyme can also be a detectable change as with the substrate, and in the sensor protein of the present invention, the change in the coenzyme may be a detectable signal.

  Preferred proteins with “detectable catalytic activity” of the present invention are those that provide a product that readily and directly generates a detectable signal. Non-limiting examples of the protein include enzymes frequently used for general enzymatic chemical detection such as alkaline phosphatase, peroxidase, and β-galactosidase. Alkaline phosphatase is an enzyme that converts p-nitrophenyl phosphate or the like into p-nitrophenol, and the activity of the enzyme is easily detected based on the characteristic color of the produced p-nitrophenol. Furthermore, since the enzyme has a relatively high stability, it is particularly useful as the protein of the present invention.

  It should be particularly noted that the term “native protein” as used herein is used only in comparison with a protein into which a circular permutation has been introduced. That is, the term “natural protein” of the present invention refers to a protein having N and C terminus (original N and C terminus) substantially the same as the “original” mature protein existing in nature. It is used only for the purpose of distinguishing from new N and C-terminal proteins produced by circular permutation. Thus, as long as it has substantially the same N and C termini as the “original” protein present in nature, it has insertions, deletions, substitutions or duplications at other sites in the protein. Mutant proteins are also included in the “natural protein” of the present invention. For example, in alkaline phosphatase derived from Escherichia coli (PhoA), it is known that a highly active mutant (D101S) can be obtained by substituting the serine residue for the 101st aspartic acid residue in the vicinity of its active site. However, such highly active mutants are also included in the “natural protein” referred to in the present invention. However, it goes without saying that a mutant in which such a mutation irreversibly loses the catalytic activity of the original protein cannot be included in the natural protein of the present invention.

In the present invention, first, the “original” N-terminus and C-terminus of the natural protein are combined by genetic engineering techniques. An appropriate “linker” is used for the binding. The linker is preferably a peptide, and it is particularly preferred that the length of the peptide is approximately equal to the distance between the natural N / C ends of the natural protein. The amino acid sequence of the peptide should be selected so as not to change the native conformation of the natural protein as much as possible. That is, the linker of the present invention should be designed so as not to have any action other than simply crosslinking the original N / C terminus of the natural protein. Such a design can be easily calculated based on the three-dimensional structure chemistry of known proteins. For example, in Escherichia coli PhoA, a linker that connects the amino N-terminal amino acid 1 and the C-terminal amino acid 449 can be designed as (G ₄ S) ₃ based on the three-dimensional structure.

  Next, in the present invention, based on the circular permutation strategy, a new N / C terminus is introduced around the catalytic active site of the natural protein in which the original N / C terminus is crosslinked with a linker as described above. As used herein, the term “catalytic active site” refers to an activity including an amino acid residue that recognizes and acts on a substrate of a natural protein and, if necessary, a coenzyme or an efactor molecule. It is an area. In addition, “peripheral catalytic active site” means that an epitope or the like of another protein is added to a position in the vicinity of the active region, preferably N / C terminal newly introduced therein by circular permutation, When a specific antibody or the like for the epitope is bound, the binding makes the substrate, coenzyme or factor inaccessible to the active region, or the activity of the active region itself can be changed. Refers to the position. Of course, if the function of the active region is irreparably damaged by the newly introduced N / C terminus, the position should be excluded from the “around the catalytic active site”. In the example of E. coli PhoA, the preferred “around the catalytically active site” may be amino acids 90-94 and the vicinity of the enzyme.

  In the sensor protein of the present invention, a “heterologous specific binding region” is present at one or both of the new N / C terminus (new N / C terminus) formed by circular permutation of the natural protein. Is added. The “heterologous specific binding region” refers to a substance comprising a substance other than the natural protein, preferably a protein or peptide itself, or a part thereof, for example, an epitope. In particular, when the heterologous specific binding region comprises an epitope of another protein / peptide, the sensor protein of the present invention can be suitably applied to a homogeneous competitive immunoassay protocol using a specific antibody against the epitope. . Further, when the specific binding region contains a peptide hormone or a receptor binding site thereof, the homogeneous assay of the present invention can be carried out using a receptor for the hormone. Non-limiting examples of heterologous specific binding regions of the present invention include regions having an epitope such as osteocalcin, insulin, serum albumin and the like. In addition, when a heterogeneous specific binding region is added to both of the new N / C ends, the peptides constituting the heterogeneous specific binding region may be the same or different at both ends. Also good. That is, when one heterologous protein / peptide has one or more epitopes, one epitope may be added to the N-terminus and another epitope may be added to the C-terminus. In addition, in addition of the heterogeneous specific binding region to the new N / C terminus, it is also a preferred embodiment that both are bound via an appropriate intervening sequence.

A preferred example of the circular permutation mutant of the present invention is a circular permutation cp90 derived from E. coli alkaline phosphatase. Moreover, as a suitable sensor protein of this invention, the fusion protein BGP2-cp90 of the said circular permutant cp90 and an osteocalcin epitope can be illustrated. cp90 is a circular permutation created based on the Escherichia coli alkaline phosphatase phoA sequence, with 90 and 94 residues from the N-terminal of phoA as new N and C-terminals. The ends are joined by a linker sequence (G ₄ S) ₃ . BGP2-cp90 is a fusion protein consisting of cp90 and the epitope comprising an osteocalcin epitope bound to the N-terminus of cp90 via Arg as an intervening amino acid residue. Of course, functional derivatives of these proteins, for example, proteins comprising an amino acid sequence having one or several amino acid deletions, additions, insertions and / or substitutions with respect to the amino acid sequence of the protein, As long as the protein comprising an amino acid sequence having homology of at least 70% or more, preferably 80% or more, more preferably 90% or more, also has the characteristic function of the protein of the present invention, the present invention Is available. The amino acid sequence homology referred to here is obtained by searching using the default parameters (matrix = Blosum62; gap existence cost = 11, gap extension cost = 1) on the Internet site http: // www. ncbi. n / m. nih. It can be defined as the percentage of positives shown by the BLASTP algorithm, which can be implemented with gov / egi-gin / BLAST. In addition, nucleic acid molecules encoding the above proteins are also useful in the production of the sensor protein of the present invention. In addition to the nucleic acid molecules specifically exemplified herein, the nucleic acid molecule herein can be encoded by many other nucleotide sequences due to the degeneracy of the genetic code, and depending on the expression system, Codons that are most frequently used in the host can be advantageously used. It goes without saying that those having these degenerate sequences are also included in the nucleic acid molecule of the present invention.

  The sensor protein of the present invention can be suitably used for a homogeneous assay. So-called homogeneous EIA, which is a homogeneous assay using an antigen-antibody reaction, is described by Rubenstein (Rubenstein, KE et al: “Homogeneous” enzyme immunoassay. A new immunochemical technique .: Biochem. Biophys. Res. Commun., 47: 846 (1972 )) Etc. Specifically, in this method, first, an antibody is bound to an enzyme-labeled antigen in which an enzyme and an antigen are bound. Usually, the binding of the antibody sterically prevents the substrate from approaching the active site of the enzyme, thereby inhibiting the activity of the enzyme. In addition to steric hindrance, the inhibition is considered to be caused by enzyme conformational changes due to antibody binding. Furthermore, in a special case, the enzyme activity of an enzyme bound to an antigen is decreased, but the enzyme activity may be recovered by binding to an antibody. In any case, in homogeneous EIA, an enzyme conjugated with an antigen is used to measure the modulation of enzyme (catalytic) activity caused by the binding of an antibody to the antigen.

In addition, from this principle, it will be possible to perform a homogeneous assay not only for antigen-antibody reaction but also for a specific protein / peptide and its specific receptor combination. In that sense, the “specific binding partner” of the present invention means that when the “heterologous specific binding region” added to the nascent N / C terminus of the present invention is an epitope of another protein / peptide, The antibody can be selected from an antibody specific to the epitope, and when the “heterologous specific binding region” is a functional peptide / protein such as a hormone, the receptor of the peptide / protein. The antibodies referred to in the present invention include polyclonal antibodies and monoclonal antibodies, as well as fragments of these antibodies, such as Fab fragments, Fab ′ fragments, F (ab ′) ₂ fragments, F (v) fragments, H chain monomers, Also included are dimers, L chain monomers or dimers, dimers composed of one H chain and one L chain. Similarly, the receptor may include a solubilized form of the receptor, a partial peptide comprising the binding region of the receptor, and a fusion protein thereof. Such various antibodies and receptors may be prepared by any method known to those skilled in the art. Further, as described above, when the same or different “heterologous specific binding region” is added to both the nascent N and C terminus, the “specific binding partner” recognizes the “heterologous specific binding region”. Can be two or more partners. Furthermore, the example where one partner recognizes both the N and C termini simultaneously and bridges it is of particular interest.

  A more specific preferred example of the homogeneous assay of the present invention can be described as a so-called competitive homogeneous immunoassay. That is, in the competitive homogeneous immunoassay of the present invention, first, a sensor protein in which an epitope of a test substance is added to its nascent N and / or C terminus is prepared, and the sample is suspected of containing the sensor protein and the test substance. Contact. The contacting is performed under conditions sufficient to form a complex of the antibody and the epitope in the presence of an antibody specific for the epitope. Thereafter, a substrate of the sensor protein of the present invention and, if necessary, a coenzyme and the like are added to the reaction product, and its catalytic activity is measured.

  In examples where antibody binding inhibits the catalytic activity of the sensor protein of the present invention, increased catalytic activity should be observed in proportion to the amount of analyte present in the sample. That is, when the test substance in the sample consumes the antibody added to the sample, the binding between the antibody and the sensor protein of the present invention is reduced, and thereby the catalytic activity of the sensor protein is restored.

  Preferably, as a control, a calibration curve may be prepared in advance by measuring a sample that does not contain the test substance or contains a known amount of the test substance. Based on the calibration curve, the test substance in the unknown sample can be quantified.

  In carrying out the homogeneous assay of the present invention, it may be preferable to supply the sensor protein of the present invention, the specific binding partner, etc. used in the assay in the form of a kit. In addition to the sensor protein of the present invention, the kit may contain a suitable buffer solution, a substrate of the sensor protein, a coenzyme, and the like in addition to the specific binding partner. The construction of such a kit and its manufacturing method will be known to those skilled in the art.

  Without further explanation, those skilled in the art given the above explanation can make full use of the present invention. The following examples are given for illustrative purposes only.

(Example 1)
Construction of Vector Introduced with Circular Permutation Two DNA fragments encoding 1-90 residues and 94-449 residues were prepared by PCR using wild-type PhoA as a template. Next, the original N / C terminus was linked via a (G ₄ S) ₃ linker sequence by overlap extension PCR, and a PCR fragment designed so that the 90th and 94th positions became the new N / C terminus was prepared. . These were inserted into pCR4-TOPO (Invitrogen) to confirm the sequence, and then incorporated into a secretory expression vector pET20b having a T7 promoter to obtain an expression vector.

(Example 1-1)
Primer design Primers for introducing circular permutation were designed and synthesized. Specifically, a circularly permuted mutant gene was prepared by linking two DNA fragments cleaved at a new N / C terminal site using linker extension PCR with a linker. Here, the following four primers were synthesized in order to produce mutants with residues 90 and 94 from the original N-terminus. The amino acid sequence of the linker was slightly longer than the original N / C terminal distance and considered to have little effect on conformation (Gly ₄ Ser) ₃ and was placed at the 5 ′ end of 1 reverse and 449 forward. Further, NotI was placed at the 5 ′ end of 94 reverse and XhoI was placed at the 5 ′ end of 90 forward as a restriction enzyme cleavage site for inserting the prepared DNA fragment.

1 reverse
5′- GATCAGGTGGAGGAGGTTCTGGAGGTGGCGGAAGT GGTACCCCAGAAATGCCTGTTC-3 ′ (SEQ ID NO: 1)
(The underlined part is the part that encodes the linker.)

449 forward
5′- CCTCCCAGAAACCTCCTCCACCTGATCCACCACCACC CCTAAGCCCCAGAGCGGCTT-3 ′ (SEQ ID NO: 2)
(The underlined part is the part that encodes the linker.)

94 reverse
5′-ATAAGAAT GCGGCCGC CGGCAAAACCGGACTACGTCAC-3 ′ (SEQ ID NO: 3)
(Underlined portion is NotI cleavage site)

90 forward
5'-CCG CTCGAG ATTCACGCGCATAGTGAGGTATTGC-3 '(SEQ ID NO: 4)
(Underlined portion is XhoI cleavage site)

(Example 1-2)
Preparation of Mutant Gene A mutant protein gene was prepared in which the original N / C ends were linked by a linker, and residues 90 and 94 were made new C / N ends. First, by PCR, a vector pET-AP (Suzuki et al., J. Biochem., Vol. 122, pp. 322-329 (1997)) encoding a wild-type phoA gene was used as a template, and Ex-Taq DNA polymerase- Two types of DNA fragments encoding 1 to 90 residues and 94 to 449 residues were prepared using Ze (Takara Bio, Kyoto). Two primers of 50 pmol each, 10 ng pET-AP, 0.2 mM dNTPs, 10 μl Ex-Taq Buffer (Takara Bio), 5 unit Ex-Taq 1 μl were mixed to make a 100 μl reaction solution. After repeating 30 cycles of 94 ° C. for 30 seconds, 56 ° C. for 30 seconds, and 72 ° C. for 1 minute and 30 seconds, the mixture was reacted at 72 ° C. for 10 minutes. The primers used at this time were as follows (Table 1).

  These two kinds of DNA fragments were electrophoresed on a 1% agarose gel containing TAE buffer, and then the gel containing the target band was cut out and purified using a QIA quick Gel extraction kit (Qiagen, Tokyo). Next, the prepared DNA fragments were joined at the linker moiety by overlap extension PCR. About 100 ng of each of the two PCR products was mixed with 0.2 mM dNTPs, 10 μl Ex-Taq Buffer, 5 unit Ex-Taq to make a 100 μl reaction solution at 94 ° C., 5 minutes later, 94 ° C. for 30 seconds, 60 ° C. After 15 cycles of 30 minutes at 72 ° C. and 1 minute and 30 seconds, the reaction was carried out at 72 ° C. for 5 minutes. The PCR product used at this time was 1-90 as PCR product1 and 94-449 as PCR product2. The target insert thus prepared was named cp90.

Thereafter, in order to increase only the target DNA, 50 pmol of two kinds of primers were added to this reaction solution in increments of 2.5 unit Ex-Taq. After 94 minutes at 94 ° C., 30 seconds at 94 ° C. After 35 cycles of 1 minute 45 seconds at 72 ° C., the reaction was carried out at 72 ° C. for 5 minutes.
The primer combination added at this time was 94 reverse as Primer 1 and 90 forward as Primer 2.

  This solution was subjected to agarose gel electrophoresis to confirm that a DNA fragment of the desired size was amplified, and the gel was cut out and purified using a QIA quick Gel extraction kit.

(Example 1-3)
Integration into Vector and Confirmation of Base Sequence The DNA solution prepared by overlap extension PCR was incorporated into a pCR4-TOPO vector (Invitrogen, Tokyo) suitable for PCR product cloning, and the base sequence was confirmed.

Using a kit using pCR4-TOPO Vector, TOPO TA Cloning Kit for Sequencing (Invitrogen) suitable for cloning and sequencing a PCR product having a 3′-dA protruding end amplified with Taq polymerase was used. An equimolar amount of PCR product was added to 1 μl of pCR4-TOPO Vector, 1 μl of Salt Solution was further added, and the mixture was allowed to stand at room temperature for 30 minutes. DH5α dissolved the Ligation Mixture on ice (Φ80d lacZ ΔM15, recA 1, endA 1, gyrA 96, thi -1, hsdR 17 (rK -, mK +), supE 44, relA 1, deoR, Δ (lacZYA - argF ) In addition to U169), left on ice for 30 minutes, and then heat shocked at 42 ° C. for 45 seconds, 200 μl of SOC medium (20 g bacto trypton, 5 g bacto yeast extract, 0.5 g NaCl, 0.2 ml 5N NaOH, 20 ml per liter) 1 M glucose, 10 ml 1 M MgCl ₂ , 10 ml 1 M MgSO ₄ ) and cured by curing at 37 ° C. for 30 minutes, transformed beforehand with 10 μl of 0.4 M IPTG and 200 μl of 10 mg / ml X-ga YT agar medium plate containing 100 μg / ml ampicillin and 1% glucose (8 g bacto trypton, 5 g bacto yeast extract, 5 g NaCl, 0.2 ml 5 N NaOH, 15 g agar per liter) at 37 ° C. Cultured overnight. On the next day, white colonies of several white colonies and blue colonies were picked with bamboo skewers, and colony PCR was performed using two primers, Ex-Taq polymerase, M13 forward, and M13 reverse. The PCR product was electrophoresed on an agarose gel, and only a colony having a band of a desired size was sterilized in a LB liquid medium containing 100 μg / ml ampicillin (10 g bacto trypton, 5 g bacto yeast extract, 5 g NaCl, 0.1 g per liter). 2 ml 5N NaOH), cultured at 37 ° C. overnight, the plasmid was extracted using Quantum Prep Plasmid Miniprep Kit (Bio-Rad, Tokyo), and the sequence was confirmed using ABI310 DNA sequencer.

Next, the sample whose sequence was confirmed was treated with a restriction enzyme and transferred to an expression plasmid vector. As an expression plasmid vector, pET20b (+) (Merck, Tokyo) that can secrete and express the target protein into the periplasm was selected. 10 unit NotI, 10 unit XhoI, 4 μl 10 × NEB Buffer 3 (Daiichi Kagaku, Tokyo), 4 μl of 0.1% BSA, with respect to 20 μl of plasmid solution in which cp90 gene was incorporated into pCR4-TOPO and pET20b (+) 11 μl of sterilized water was added and treated at 37 ° C. for 16 hours. After restriction enzyme treatment, 2 μl of the restriction enzyme-treated and excised and purified vector and 5 μl of Ligation high (Toyobo, Osaka) were added to 3 μl of the inserted DNA fragment excised and purified by agarose gel electrophoresis, and ligation reaction was performed at 16 ° C. for 30 minutes. Went. Among using ^{5μl XL10-Gold (Tet r,} Δ (mcrA) 183, Δ (mcrCB - hsdSMR - mrr) 173, endA 1, supE 44, thi -1, recA 1, gyrA 96, relA, lac, The , [F ′, proAB , lacl ^q Z ΔM15, Tn10 (Tet ^r ), Tn5 (Kan ^r ), Amy], were transformed with 37 μl on a YT agar medium plate containing 100 μg / ml ampicillin and 1% glucose. Cultivated overnight at 0 ° C. On the next day, colonies were picked with bamboo skewers, inoculated into 5 ml of LB liquid medium containing 100 μg / ml ampicillin, cultured overnight at 37 ° C., and cultured with Quantum Prep Plasmid Miniprep Kit for pET20-cp90. Plasmid vector Out was.

(Example 1-4)
Expression of proteins, purified pET20b (+) to using 5μl of the vector pET20-cp90 incorporating BL21 (DE3, pLysS) (F- , ompT, hsdS B, (r B -, m B -), dcm, gal , Λ (DE3), pLysS, Cm ^r ) were transformed and cultured overnight at 37 ° C. on a YT agar plate containing 100 μg / ml ampicillin and 1% glucose. The next day, colonies were picked on bamboo skewers, inoculated into 5 ml of LB liquid medium containing 100 μg / ml ampicillin, cultured overnight at 37 ° C., then inoculated into 250 ml of LB liquid medium containing 100 μg / ml ampicillin, and cultured at 27 ° C. O. The 1M IPTG when the D ₆₀₀ reached about 0.6 to induce expression by adding 25 [mu] l, and incubated further for 5-6 hours at 27 ° C.. Thereafter, the culture solution is centrifuged at 6000 G for 10 minutes at 4 ° C., the supernatant is discarded, and the cells are resuspended in 30 ml of Sonication Buffer (50 mM NaH ₂ PO ₄ , 10 mM Tris-HCl, 100 mM NaCl, pH 8.0). The suspension was transferred to a 50 ml tube and centrifuged again at 6000 G for 10 minutes at 4 ° C. After discarding the supernatant, the cells were resuspended with 10 ml Sonication Buffer and transferred to a 15 ml tube. After reducing the volume in this way, the cells were crushed with Digital Sonifier (BRANSON) and centrifuged at 9000 G at 4 ° C. for 1 hour. 300 μl of TALON metal affinity column (Clontech, Tokyo) was added to the supernatant, and the protein was purified by a batch / gravity column purification method. Elution Buffer (50 mM NaH ₂ PO ₄ , 20 mM MES, 100 mM NaCl, pH 5.0) was used for protein elution. Since NaH ₂ PO ₄ inhibits enzyme activity, a buffer for storage (50 mM Tris-HCl, 150 mM) was used using Amicon Ultra-15 Centrifugal Filter Devices (Millipore, Tokyo) so that the concentration of NaH ₂ PO ₄ was 1 nM or less. The buffer was replaced with NaCl, pH 8.0).

(Example 1-5)
Activity measurement First, the protein concentration was measured by Bradford protein assay (Bio-Rad). P-Nitrophenylphosphate ditrit salt (Sigma) as a substrate was dissolved in Buffer (1M Tris-HCl, 10 mM MgSO ₄ , 50 μM ZnSO ₄ ) for activity measurement to obtain a substrate solution. 5 μl of enzyme solution was added to 95 μl of substrate solution of various concentrations, and the change in absorbance was measured using a 96-well microplate. The reaction rate for each concentration was measured, and Vmax and Km were determined from Hanes-Woolf plot.

(Example 1-6)
In order to confirm whether cp90 in which the confirmation activity of cp90 was recognized was correctly prepared, SDS-PAGE, N-terminal peptide sequence, and far ultraviolet CD spectrum measurement were performed.

(Example 1-7)
As a result , activity was confirmed in the expressed and purified cp90. 3 and 4 show the relationship between the substrate concentration and the reaction rate in WT-AP (wild type) and cp90 (circular permutant). Hanes-Woolf plot (FIGS. 5 and 6) was performed to determine Vmax and Km. The results are shown in Table 2 below.

  A migration photograph of SDS-PAGE of WT-AP and cp90 is shown in FIG. A band was confirmed at almost the same position as WT-AP. The band is seen at a slightly longer position than WT-AP because it is longer by the linker. In the N-terminal peptide sequence, 25 residues from the N-terminal were read, and the following sequence was obtained. As a result, it was confirmed that the underlined portion was the 94th to 98th residue sequence in cp90. The N-terminal 20 residues coincided with the sequence derived from the multicloning site of the vector. It was also confirmed that the PelB signal sequence encoded on the N-terminal side was cleaved.

N-terminal peptide sequence result N′-MDIGINSDPNSSSSVDKLAAA GKPDY- C ′ (SEQ ID NO: 5)

  The result of the far ultraviolet CD spectrum measurement of WT-AP and cp90 is shown in FIG. It was confirmed that cp90 has almost the same secondary structure content as WT-AP.

( Example 2)
The substitution of osteocalcin epitope insertion cp90N end of osteocalcin epitope was carried out as follows.

(Example 2-1)
Primer design and insert preparation cp90 was Template, and a primer for introducing DNA encoding an anti-osteocalcin antibody recognition site (epitope) into the 5 'end was designed by PCR. At this time, in order to remove the multicloning site-derived sequence present at the N-terminus of cp90, NcoI was placed as a restriction enzyme cleavage site at the 5′-end of the primer. In addition, if the osteocalcin epitope and the 94th residue are directly bound, it is considered that the inserted epitope is buried in alkaline phosphatase and the antigen-antibody reaction may be inhibited. Therefore, the osteocalcin epitope C is used as a spacer. Two types of primers were prepared, one that encodes one terminal residue Arg and one that does not.

  A DNA fragment in which the osteocalcin antibody recognition site coding region was inserted at the 5 'end was prepared by PCR using cp90 as a template and the primers BGP1, BGP2 and 90 forward. The combinations of the prepared DNA fragment names and primers are as follows (Table 3).

  A part of this PCR product was subjected to agarose gel electrophoresis, and it was confirmed that a DNA fragment of the desired size was prepared. To these PCR products, NcoIl l, 10x NEB Buffer (BIO RAD) 2l, and MilliQ water 17l were added and treated with a restriction enzyme at 37 ° C for 16 hours. For pET20b-cp90 as a vector into which a DNA fragment was incorporated, a similar enzyme solution was added, and 10 unit CIAP (Takara Bio) was further added to prevent self-ligation and left at 37 ° C. for 16 hours. Thereafter, the gel was cut out and purified.

(Example 2-2)
Construction of vector and confirmation of sequence of insert The DNA fragment purified by treatment with restriction enzyme and the expression vector plasmid were ligated by a conventional method. Using this ligation mixture, XL10-Gold was transformed and cultured overnight at 37 ° C. on a YT agar medium plate containing 100 μg / ml ampicillin and 1% glucose. In order to confirm whether or not DNA fragments treated with NcoI at both ends did not enter in the reverse direction, colonies were picked with bamboo skewers, and colony PCR was performed using T7 primer and 407 forward primer. If the insert is correctly inserted, a band should appear around 230 bp. The PCR product was subjected to agarose gel electrophoresis, only a colony with a band near 230 bp was picked with bamboo skewers, inoculated into 5 ml of LB liquid medium containing 100 μg / ml ampicillin, cultured at 37 ° C. overnight, Quantum Prep Plasmid Miniprep Two types of vectors, pET20-BGP1cp90 and pET20-BGP2cp90, in which an osteocalcin epitope was inserted at the N-terminus of cp90, were constructed using Kit.

  Sequencing was performed to confirm that this vector had the correct sequence. Since the concentration of the extracted plasmid was low, PCR was performed by Ex-Taq using T7 primer and 407 forward primer to amplify the DNA fragment to be read. To the reaction product, 5 times the amount of QG Buffer (Qiagen) and an equivalent amount of isopropanol were added, stirred, transferred to a column of QIA quick Gel extraction kit (Qiagen), and centrifuged. Thereafter, similarly to gel cutting and purification, the cells were washed once with 500 μl of QG Buffer and 750 μl of PE Buffer and then eluted with EB Buffer. The DNA fragment thus purified was sequenced using T7 primer.

(Example 2-3)
Expression and purification of protein BL21 (DE3, pLysS) was transformed with two types of vectors pET20-BGP1cp90 and pET20-BGP2cp90 constructed in the above “2-2”, and 100 μg / ml ampicillin, 37 μg / ml chloram The cells were cultured overnight at 37 ° C. on a YT agar plate containing phenicol and 1% glucose. The next day, the colonies were picked with bamboo skewers, inoculated into 5 ml of LB liquid medium containing 100 μg / ml ampicillin and 34 μg / ml chloramphenicol, cultured at 37 ° C. overnight, then 100 μg / ml ampicillin, 34 μg / ml chloram. Inoculated into 250 ml of LB liquid medium containing phenicol, cultured at 27 ° C. To induce the expression by adding 25μl of 1M IPTG when the D ₆₀₀ reached about 0.4 to 0.6, and further cultured for 8 hours at 27 ° C.. Thereafter, the culture solution is centrifuged at 6000 G for 10 minutes at 4 ° C., the supernatant is discarded, the cells are resuspended in 30 ml Sonication Buffer, transferred to a 50 ml tube, and again centrifuged at 6000 G for 4 minutes at 4 ° C. After discarding the cells, the cells were resuspended with 10 ml Sonication Buffer and transferred to a 15 ml tube. After reducing the volume in this way, the cells were crushed with Digital Sonifier and centrifuged at 9000 G for 1 hour at 4 ° C. Thereafter, a TALON metal affinity column was added to the supernatant, and the protein was purified by a batch / gravity column purification method. An elution buffer was used for protein elution.

(Example 2-4)
Activity measurement First, the protein concentration was measured by Bradford protein assay. P-Nitrophenylphosphate ditrit salt as a substrate is dissolved in Buffer (1M Tris-HCl, 10 mM MgSO ₄ , 50 μM ZnSO ₄ ) for activity measurement to obtain a substrate solution. 5 μl of enzyme solution was added to 95 μl of substrate solution of various concentrations, and the change in absorbance was measured using a 96-well microplate. The reaction rate for each concentration was measured, and Vmax and Km were determined from Hanes-Woolf plot.

(Example 2-5)
Binding of BGP2-cp90 to anti-BGP (osteocalcin) antibody Solid-phase enzyme immunoassay (ELISA) to determine whether circular permutant BGP2-cp90 having a BGP epitope at the N-terminus can bind to anti-BGP (osteocalcin) antibody ). Mouse anti-osteocalcin antibody (Mitsubishi Chemical Yatron, Tokyo) diluted to 5 μg / ml in PBS (10 mM phosphate, 145 mM NaCl, 5 mM KCl, pH 7.2) or anti-His-tag antibody diluted to 0.2 μg / ml ( Penta-His, Invitrogen) or PBS was dispensed in 100 μl portions onto a Falcon 3912 microplate and placed at 4 ° C. for 16 hours. To this was added PBS containing 1% skim milk (MPBS), left at 25 ° C. for 2 hours, washed with PBS containing 0.1% Tween-20 (PBS-T), and then purified with 2 μg / ml of purified BGP2- MPBS with or without cp90 was added and placed at 37 ° C. for 1 hour. In order to detect the alkaline phosphatase solid-phased so far, rabbit anti-Escherichia coli alkaline phosphatase IgG (Rockland Inc.) diluted 1/1000 with MPBS was added and placed at 25 ° C. for 1 hour, and three times with PBS-T. After washing twice, horseradish peroxidase-labeled anti-rabbit IgG diluted to 1/5000 with MPBS was added and left at 25 ° C. for 1 hour. The plate was washed three times with PBS-T, and a reaction was started by adding 100 μl of an enzyme reaction solution (50 ml ELISA buffer, TMBZ (in DMSO) 500 μl, H ₂ O ₂ 10 μl) prepared in advance to each well. After reacting in the dark for 15 minutes, 50 μl of 3.2N H ₂ SO ₄ was added to stop the reaction, and the absorbance was measured with a plate reader (450 nm, control was 655 nm).

(Example 2-6)
Activity change in the presence of a specific antibody in BGP2-cp90 Furthermore, in order to investigate how various antibodies affect the activity of the prepared enzyme, a mouse anti-osteocalcin antibody or purified mouse myeloma IgM ( Zymed, San Francisco, Calif.) Was added in an amount of 5 μg, and the enzyme activity of each prepared to a total amount of 100 μl was measured in the same manner as in “2-4 Activity measurement” above.

(Example 2-7)
Results The protein could be expressed and purified in both BGP1-cp90 and BGP2-cp90, and the activity was also recognized. The result of the enzyme activity measurement about BGP2-cp90 is shown in FIG.

  Further, the result of specific binding of BGP2-cp90 to the anti-BGP (osteocalcin) antibody is shown in FIG. FIG. 10 shows that BGP2-cp90 markedly changes the amount of binding to the microplate depending on whether or not the anti-osteocalcin antibody is immobilized, as in the case of immobilizing the antibody against the His6 tag located at the BGP2-cp90 C-terminus. I understood. In addition, the measured value when BGP2-cp90 was not added was small when either antibody was immobilized, and it was found that the non-specific adsorption of the anti-osteocalcin antibody was small. That is, from this experiment, it was shown that the anti-osteocalcin antibody can specifically bind to the N-terminal tag of BGP2-cp90.

  Finally, in BGP2-cp90, an anti-osteocalcin antibody-specific activity change was observed. The relationship between the substrate concentration and reaction rate in BGP2-cp90 is shown in FIGS. Based on this, Hanes-Woolf plot (FIG. 13) was performed to obtain Vmax and Km. The results are shown in Table 4 below.

  From these results, it was shown that the sensor protein of the present invention can be effectively used in a homogeneous assay.

  The base sequence and amino acid sequence of the circular permutation cp90 of E. coli phoA and the fusion protein BGP2-cp90 of the protein and osteocalcin epitope are shown below.

  The present invention provides a new technique for preparing hybrid proteins by introducing specific binding regions derived from various heterologous proteins around the active site of a sensor protein. The catalytic activity of the sensor protein thus obtained is modulated based on the interaction between the specific binding region derived from the heterologous protein introduced therein and the specific binding partner for the region. Based on this modulation, a very specific and sensitive biochemical assay is achieved.

FIG. 1 shows the crystal structure of alkaline phosphatase (dimer). FIG. 2 schematically shows the DNA structure of alkaline phosphatase cp90 (+ His tag) into which a circular permutation has been introduced. FIG. 3 shows the relationship between the substrate concentration and the reaction rate in WT-AP (wild type alkaline phosphatase). FIG. 4 shows the relationship between the substrate concentration and the reaction rate in cp90 (alkaline phosphatase circular permutant). FIG. 5 shows a WT-AP Hanes-Woolf plot. FIG. 6 shows a Hanes-Woolf plot of cp90. FIG. 7 is a diagram illustrating a situation of SDS-PAGE of WT-AP and cp90. FIG. 8 shows the far-UV CD spectrum measurement results of WT-AP and cp90. FIG. 9 shows the relationship between the substrate concentration and the reaction rate in the sensor protein BGP2-cp90 of the present invention. FIG. 10 shows specific binding of anti-BGP (osteocalcin) antibody to BGP2-cp90. The results represent the average and standard deviation of 3 samples. FIG. 11 shows the relationship between the substrate concentration of the sensor protein BGP2-cp90 of the present invention and the reaction rate in the presence of an anti-BGP antibody. FIG. 12 shows the relationship between the substrate concentration of the sensor protein BGP2-cp90 of the present invention and the reaction rate in the presence of mouse myeloma IgM antibody. FIG. 13 shows Hanes-Woolf plots of BGP2-cp90, BGP2-cp90 + anti-BGP antibody and BGP2-cp90 + mouse myeloma IgM antibody.

Claims (19)

  The native N-terminal and C-terminal of a natural protein having a detectable catalytic activity are linked by a linker to generate new N-terminal and C-terminal around the catalytic active site of the protein, and the new N-terminal and A sensor protein produced by binding a heterogeneous specific binding region to the C-terminus.   The sensor protein according to claim 1, wherein the natural protein having a detectable catalytic activity is an enzyme.   The sensor protein according to claim 2, wherein the enzyme is alkaline phosphatase.   The sensor protein according to any one of claims 1 to 3, wherein the heterologous specific binding region is an epitope of another protein.   The sensor protein according to claim 4, wherein the epitope of the other protein is an epitope of osteocalcin. A homogeneous assay method for identifying and / or quantifying the presence of a test substance in a sample, the assay method comprising:
(1) The original N-terminal and C-terminal of a natural protein having a detectable catalytic activity are linked by a linker, and new N-terminal and C-terminal are generated around the catalytic active site of the protein. Using a sensor protein produced by binding a heterogeneous specific binding region derived from a test substance to the N-terminus and / or C-terminus,
(2) contacting a sample presumed to contain a test substance with the sensor protein in the presence of at least one specific binding partner for the heterogeneous specific binding region; and (3) a sensor by the contact Measuring the modulation of the catalytic activity of the protein,
The homogeneous assay method comprising:   The method of claim 6, wherein the homogeneous assay is a competitive assay.   The method according to claim 6 or 7, wherein the natural protein having a detectable catalytic activity is an enzyme.   9. The method of claim 8, wherein the enzyme is alkaline phosphatase.   The method according to any one of claims 6 to 9, wherein the heterologous specific binding region is an epitope of another protein, and the specific binding partner is an antibody against the epitope.   The method according to claim 10, wherein the epitope of the other protein is an epitope of osteocalcin.   A kit for homogeneous assay comprising the sensor protein of claim 1 and a binding partner specific for a heterogeneous specific binding region on the protein.   The kit according to claim 12, wherein the heterologous specific binding region is an epitope of another protein, and the specific binding partner is an antibody against the epitope. (1) consisting of the amino acid sequence represented by SEQ ID NO: 9, or (2) from an amino acid sequence having a deletion, addition, insertion and / or substitution of one or several amino acids relative to SEQ ID NO: 9. And having alkaline phosphatase activity,
A circularly permuted protein of E. coli alkaline phosphatase.   A nucleic acid molecule encoding the protein of claim 14.   The nucleic acid molecule according to claim 15, which is represented by SEQ ID NO: 8. (1) consisting of the amino acid sequence represented by SEQ ID NO: 11, or (2) from an amino acid sequence having a deletion, addition, insertion and / or substitution of one or several amino acids relative to SEQ ID NO: 11. And having alkaline phosphatase activity,
E. coli alkaline phosphatase circular permutant and osteocalcin epitope fusion protein.   A nucleic acid molecule encoding the protein of claim 17.   The nucleic acid molecule according to claim 18, which is represented by SEQ ID NO: 10.

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