WO2005063964A1 - Fkbp type ppiase, expression vector, transformant, fused protein, method of producing fused protein, method of producing target protein, method of purifying kfbp type ppiase and method of purifying fused protein - Google Patents

Abstract

It is intended to produce a target protein at a high efficiency with the use of the action of FKBP type PPIase which shows a sufficient activity in a region from low temperature to ordinary temperature in usual biochemical environment. Owing to the action of FKBP type PPIase originating in a non-thermophilic archaebacterium belonging to methane-producing bacteria, a target protein is obtained as a normal protein. By a fused protein composed of FKBP type PPIase with the target protein or the coexpression of the FKBP type PPIase with the target protein, the target protein can be properly folded. By using a vector which can express the fused protein, the target protein can be produced at a high efficiency.

Description

 Specification

 FKBP-type PPIase, expression vector, transformant, fusion protein, method for producing fusion protein, method for producing target protein, method for purifying FKBP-type PPIase, and method for purifying fusion protein

 Technical field

 The present invention relates to a method for producing an FKBP-type PPIase, an expression vector, a transformant, a fusion protein, a fusion protein, a method for producing a target protein, a method for purifying an FKBP-type PPIase, and a method for purifying a fusion protein. More specifically, an FKBP-type PPIase exhibiting sufficient activity in a low-temperature to normal-temperature range and under a normal biochemical environment, an expression vector capable of expressing a fusion protein of the FKBP-type PPIase and a target protein, A transformant containing the expression vector, a fusion protein of FKBP-type PPIase and the target protein and a method for producing the same, a method for producing the target protein using the FKBP-type PPIase, and a method for producing the PPIase by hydrophobic interaction chromatography. The present invention relates to a purification method and a method for purifying the fusion protein by hydrophobic interaction chromatography.

 Background art

 [0002] In recent years, the genome analysis of various organisms is being completed, and it is considered that future research will proceed to comprehensive functional analysis of proteins that are gene expression products. In other words, research is evolving rapidly to clarify the properties of individual proteins and comprehensively analyze the interactions between proteins to help elucidate life phenomena. In particular, intracellular receptor proteins that specifically bind to various physiologically active substances and transmit their actions are three-dimensional because the active substance that binds to the receptor protein can be a candidate for a new drug. There is significant interest in structural determination.

[0003] When determining the properties of a protein, the protein is often synthesized in large quantities by recombinant DNA technology and used as an analysis sample. That is, a gene encoding a target protein is incorporated into an expression vector, the expression vector is introduced into a host such as a bacterium, yeast, or insect cell, and the gene for the target protein is expressed in the host. Generally, a method of examining the properties of However, the nature of the protein In order to evaluate the protein, not only the primary structure but also the three-dimensional structure is extremely important. In the recombinant protein obtained as described above, the folding reaction is not performed correctly in the host, and the In many cases, the protein does not have a three-dimensional structure and cannot be obtained as an abnormal protein. Furthermore, such abnormal proteins are often obtained as insoluble aggregates called inclusion bodies rather than soluble ones. For example, when the target protein is an antibody, it is known that, when Escherichia coli is used as a host, most of the synthesized recombinant proteins are insoluble inclusion bodies (Non-patent Document 1). In addition, when a protein originally intended to be buried on the surface or inside of a biological membrane, such as an intracellular receptor protein, is a target protein, expression of the protein as a recombinant protein results in inclusion bodies. Prone.

 [0004] In some cases, the recombinant protein is degraded by the protease of the host cell, and the target protein to be analyzed is hardly obtained. Furthermore, when the target protein is toxic to cells, the recombinant protein may not even be expressed.

 [0005] As a method for refolding the abnormal protein that has become an inclusion body and returning it to a correct three-dimensional structure, the inclusion body is dissolved with a high concentration of guanidine hydrochloride, urea, or the like, and then an appropriate buffer solution is added. For example, a method of diluting to about 30 to 100 times with such a method is known. For example, Patent Literature 1 discloses that abnormal proteins are correctly folded by including a chaperonin having a function of promoting protein folding in a diluted buffer solution containing an enclosure, so that a target protein can be converted to a normal protein. The method of acquiring as is described. However, it is more difficult than expected to refold the abnormal protein that has become an encapsulated protein and return it to the correct three-dimensional structure. In addition, shadinin requires a nucleotide triphosphate such as ATP, which is a high energy substance, during the folding reaction, which is economically disadvantageous. In addition, Shachinin is a huge molecule having a molecular weight of 800,000 to 1,000,000, so that it needs an extremely large amount by weight to be used in the folding reaction, which is also economically disadvantageous.

[0006] In addition, the target protein is converted into a normal protein by the action of daltathione-S-transferase (GST), thioredoxin, maltose binding protein (MBP), NusA, and the like. It has been suggested how to obtain it, but the effect is not enough.

 [0007] On the other hand, several methods aimed at improving the yield of expression products have been disclosed. Patent Literature 2 discloses a method for increasing the yield of a target protein by fusing a partial fragment of chapanin, which is one of molecular chaperones, with a target protein (target polypeptide). . Furthermore, it is disclosed that, if a signal peptide is added to a target protein and secreted and expressed, for example, in the periplasmic region of Escherichia coli, the target protein, which is usually an inclusion body, can be expressed as a soluble protein. However, when expressed in the periplasmic region, not only the yield of the expression product is low! /, But also the solubility itself is insufficient.

[0008] Peptidyl loop mouth lys cis trans isomerase (Peptidyl-prolyl cis-trans isomerase ₀ or less, referred to as "PPIase") is one of the folding factors involved in protein folding, Among the amino acids in the protein under folding, it has the activity of catalyzing the cis-trans isomerization reaction of the peptide bond at the N-terminal side of the proline residue (PPIase activity). It is known that FKBP (FK506 Binding Protein) -type PPIase derived from thermophilic archaea or hyperthermophilic archaea having an optimum growth temperature of 55 ° C or more has not only PPIase activity but also molecular chaperone activity. Have been. Here, “molecular chaperone activity” is defined as an activity that suppresses irreversible aggregation of proteins and promotes refolding of denatured proteins. And, a method of obtaining a normal protein using FKBP-type PPIase having this molecular chaperone activity has been proposed (Non-Patent Document 2, Non-Patent Document 3, Non-Patent Document 4, and Non-Patent Document 5). The molecular chaperone activity of this FKBP-type PPIase derived from thermophilic or hyperthermophilic archaebacteria differs from the molecular chaperone activity of chaperonin, and its expression does not require a high energy substance such as ATP. , Excellent in point.

 Patent Document 1: Japanese Patent Application Laid-Open No. Hei 9 220092

 Patent document 2: International publication 00Z075346 pamphlet

 Non-patent Document 1: Pluckthun, Biotechnology, Vol. 9, pp. 545, 1991

Non-Patent Document 2: Furutani, Biochemistry, Vol. 39, No. 4 Page 53, 2000

 Non-Patent Document 3: Ideno, Eurobian's Journal of Biochemistry. (Eur. J. Biochem.), Vol. 267, No. 3139, 2000

 Non-patent Document 4: Ideno, Biochemical 'Journal (Biochem. J.), Vol. 357, pp. 465, 2001

 Non-Patent Document 5: Ideno, Applied Environmental Microbiol., Appl. Env. Microbiol. 68, 464—2002

 Disclosure of the invention

 Problems to be solved by the invention

[0010] Usually, when handling proteins, they are often handled in a low to normal temperature range, specifically, a temperature range of about 4-137 ° C to avoid heat inactivation. When a recombinant protein is produced using a host such as Escherichia coli or yeast, the protein is often produced at the optimal growth temperature of the host at 25 to 37 ° C. However, the optimal growth temperature of the above-mentioned thermophilic archaea or hyperthermophilic archaea is 55 ° C or higher, and the temperature at which the maximum activity of FKBP-type PPIase derived from these archaea is considered to be 55 ° C or higher. . Therefore, these FKBP-type PPIases are unsuitable for correctly folding the target protein in a test tube or host where the activity is high in the low to normal temperature range. On the other hand, extremely halophilic bacteria are known as archaea at room temperature. In other words, since moderately halophilic bacteria have an optimum growth temperature range around 37 ° C, FKBP-type PPIase derived from the bacteria is expected to have high activity from low to normal temperature. However, since the inside of the cells of highly halophilic bacteria is maintained at a high salt concentration of about 13M, the proteins of the highly halophilic bacteria often show activity only at high salt concentrations. It is not known whether it shows sufficient activity under normal biochemical conditions. On the other hand, methanogens having methanogenic ability are known as other normal-temperature archaebacteria. However, FKBP-type PPIase derived from methanogens has not yet been isolated, and its action and physical properties are uncertain.

[0011] An object of the present invention is to provide an FKBP-type PPIase exhibiting sufficient activity even in a low-temperature to normal-temperature range and in a normal biochemical environment. Expression vector capable of expressing a fusion protein of , A method for producing a fusion protein of FKBP-type PPIase and a target protein, a method for producing a target protein using the FKBP-type PPIase, a method for purifying the PPIase, and a method for purifying the fusion protein. Provision is also an object of the present invention. Means for solving the problem

As a result of intensive studies, the present inventors isolated FKB P-type PPIase derived from a mesophilic archaea belonging to methanogens, and the FKBP-type PPIase has molecular chaperone activity, It has been found that it shows sufficient activity both in the region and under normal biochemical environment. Furthermore, it has been found that among the FKBP-type PPIases, those having a specific amino acid sequence, and particularly those having no specific amino acid sequence deleted or originally possessed, have extremely high molecular chaperone activity. Furthermore, they have found that the target protein can be correctly folded with extremely high efficiency by allowing the FKBP-type PPIase to act on the target protein, and completed the present invention. That is, the gist of the present invention is as follows.

 [0013] (1) FKBP-type PPI ase ^ characterized by being derived from a mesophilic archaea belonging to methanogens

 (2) The FKBP-type PPIase according to (1), wherein the room temperature archaea is a Methanosarcina archaea.

 (3) FKBP-type PPIase according to (1) or (2), which is a short-type FKBP-type PPIase.

 (4) The FKBP according to (1), (2) or (3), which has an amino acid sequence containing a part of the motif having the amino acid sequence represented by SEQ ID NO: 1, 2 or 3. Type PPIase,

(5) The method according to (1), (2), (3) or (4), wherein the motif comprising the amino acid sequence represented by SEQ ID NO: 4 has an amino acid sequence that is not contained in its N-terminal region. PPI ase ^ listed

 (6) an FKBP-type PPIase, which has the amino acid sequence of SEQ ID NO: 6,

(7) FKBP-type PPIase, which has the amino acid sequence of SEQ ID NO: 8,

(8) an FKBP-type PPIase, which has the amino acid sequence of SEQ ID NO: 10,

(9) FKBP according to (1) or (2), which is a long type FKBP type PPIase Type PPIase,

 (10) an FKBP-type PPIase having the amino acid sequence of SEQ ID NO: 18

(11) FKBP-type P Plase described in (1), (2), (3), (4), (5), (6), (7), (8), (9) or (10) and substantially An FKBP-type PPIase having substantially the same amino acid sequence and substantially the same activity.

 Furthermore,

 (12) FK BP according to (1), (2), (3), (4), (5), (6), (7), (8), (9), (10) or (11) A first coding region encoding a type PPIase, and at least one restriction enzyme site, wherein the second coding region encoding the target protein is inserted into the restriction enzyme site. An expression vector characterized by being capable of expressing the fusion protein;

 (13) The expression vector according to (12), wherein the restriction enzyme site is located downstream of the first coding region.

 (14) The expression vector according to (12) or (13), wherein the expression vector has a nucleotide sequence encoding a peptide linker between the first coding region and the restriction enzyme site.

 (15) The expression vector according to (14), wherein the peptide linker comprises a protease-digested amino acid sequence.

 (16) An expression vector characterized in that a second coding region encoding a target protein is incorporated into a restriction enzyme site of the expression vector according to (12), (13), (14) or (15). Vector

 (17) The expression vector according to (16), wherein the second coding region is a gene encoding an antibody or a partial fragment of the antibody.

 (18) The expression vector according to (16), wherein the second coding region is a gene encoding an intracellular receptor protein.

 (19) A transformant comprising the expression vector according to (16), (17) or (18),

(20) The transformant according to (19), wherein the host is Escherichia coli. further,

 (21) FK BP according to (1), (2), (3), (4), (5), (6), (7), (8), (9), (10) or (11) Fusion protein of type PPIase and target protein,

 (22) The fusion protein according to (21), further comprising a protease-digested amino acid sequence between the FKBP-type PPIase and the target protein.

 (23) A fusion protein characterized in that the first and second coding regions incorporated in the expression vector described in (16) are transcribed and translated to synthesize a fusion protein of FKBP-type PPIase and a target protein. Production method,

 (24) The first coding region and the second coding region incorporated in the expression vector described in (16) are transcribed and translated to synthesize a fusion protein of FKBP-type PPIase and a target protein, and then, the target protein is A method for producing a target protein,

 (25) the expression vector has a nucleotide sequence encoding a protease-digested amino acid sequence between the first coding region and the second coding region, the first coding region, a nucleotide sequence encoding a protease-digested amino acid sequence, By transcribing and translating the second coding region, synthesizing a fusion protein having a protease-digested amino acid sequence between FKBP-type PP Iase and the target protein, and allowing the protease to act on the fusion protein, the target protein is converted. The method for producing a target protein according to (24), which is characterized by cutting out,

 (26) FK BP according to (1), (2), (3), (4), (5), (6), (7), (8), (9), (10) or (11) By co-expressing the gene encoding type PPIase and the gene encoding the target protein in the same host, FKBP-type PPIase acts on the target protein in the host, solubilizing the target protein and dissolving the host. A method for producing a target protein, which comprises collecting the target protein from the fraction;

 (27) FK BP according to (1), (2), (3), (4), (5), (6), (7), (8), (9), (10) or (11) A method for purifying an FKBP-type PPIase, comprising removing a component other than the FKBP-type PPIase by subjecting a sample containing the type-PPIase to hydrophobic interaction chromatography,

(28) FK described in (1), (2), (3), (4), (5), (6), (7), (8), (9), (10) or (11) A method for purifying a fusion protein, comprising subjecting a sample containing a fusion protein of a BP-type PPIase and a target protein to hydrophobic interaction chromatography to remove components other than the fusion protein.

 The invention's effect

 [0016] According to the FKBP-type PPIase of the present invention, the target protein can be efficiently folded even in a low to normal temperature range and under a normal biochemical environment.

[0017] According to the expression vector of the present invention, a fusion protein of FKBP-type PPIase and a target protein can be expressed, and the target protein can be expressed in a low to normal temperature range and in a normal biochemical environment. Folds efficiently.

According to the transformant of the present invention, it is possible to produce a fusion protein of FKBP-type PPIase and a target protein exhibiting sufficient activity even in a low-temperature to normal temperature range and under a normal biochemical environment.

 [0019] According to the fusion protein of the present invention, the target protein is efficiently folded in a low-temperature to normal-temperature range and under a normal biochemical environment.

According to the method for producing a fusion protein of the present invention, the fusion protein of the present invention can be produced efficiently.

 According to the method for producing a target protein of the present invention, a correctly folded target protein can be produced with high efficiency.

[0022] According to the method for purifying FKBP-type PPIase of the present invention, FKBP-type PPIase exhibiting high activity in a low-temperature to normal-temperature range and under a normal biochemical environment can be easily purified.

According to the method for purifying a fusion protein of the present invention, a fusion protein useful for producing a target protein can be easily purified.

 Brief Description of Drawings

 FIG. 1 is a diagram in which amino acid sequences of nine short-type FKBP-type PPIases derived from archaea belonging to methanogens are arranged and compared.

 FIG. 2 is a schematic diagram showing the configuration of the expression vector pMml8F2 constructed in Example 1.

[FIG. 3] SDS-PAGEZCBB gel showing the result of producing MaFKBP17.8 in Example 5. It is a photograph of Le.

 FIG. 4 is a photograph of a gel of SDS-PAGE / CBB showing the result of producing MaFKBP28.0 in Example 8.

 FIG. 5 is a graph showing the results of evaluating the molecular chaperone activity of MaFKBP17.8 performed in Example 9.

 FIG. 6 is a photograph of a gel of SDS-PAGEZCBB in Example 10, where FIG. 6 (a) shows the result of producing a fusion protein, and FIG. 6 (b) shows the result of producing rhodanese. 6 (c) shows the result of the comparative example.

 FIG. 7 is a photograph of a gel of SDS-PAGEZCBB in Examples 15, 16 and 17, wherein FIG. 7 (a) shows the result of producing the fusion protein in Example 15, and FIG. FIG. 7 shows the result of producing the fusion protein, and FIG. 7 (c) shows the result of producing the fusion protein in Example 17.

 BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the best mode for carrying out the present invention will be described in detail.

[0026] One of the FKBP-type PPIases of the present invention is derived from a mesophilic archaeon belonging to methanogens. Here, “room temperature archaea” refers to species belonging to archaea among normal temperature bacteria (also called mesophilic bacteria). According to the classification method of Madigan et al., Generally, the optimum growth temperature is 20-45 ° C. (Madigan, MT et al., 1997, "Block Biology of Microorganizm 8th ed., Prentice Hall, New York".) In this case, the "optimum growth temperature" refers to the microorganism. Refers to the temperature at which the growth rate of the cells is the fastest. The mesophilic archaea belonging to the methanotroph are not particularly limited, and include, for example, archaea belonging to the order Methanosa rcma, Methanobacterium J¾, Methanosphaera rot, Methanococcus, Methanocaldococcus, MethanoignisJU, and MethanomicroDiaies. Can be In particular, archaea belonging to the genus Methanosarcina, in particular, FKBP-type PPIases derived from Mathanosarcma barkeri, Methanosarcma mazei, and Methanosarcm acetivorans, exhibit PPIase activity and activity at low to normal temperatures and in normal biochemical environments. It is preferred that Z or molecular chaperone activity is high. The protein folding action of the FKBP-type PPIase of the present invention depends on its molecular The PPIase activity also contributes, which is considered to be largely due to the activity of the enzyme.

[0027] In general, FKBP-type PPIases are roughly classified into short-type FKBP-type PPIases having a molecular weight of about 16 to 18 kDa and long-type FKBP-type PPIases having a molecular weight of about 26 to 33 kDa. Analysis of FKBP-type PPIases shows that both short-type and long-type PPIases have molecular chaperone activity.It is known that short-type FKBP-type PPIase has higher PPIase activity. . Furthermore, the archaebacterial FKBP-type PPIase has an FKBP domain responsible for PPIase activity and FK506 binding activity in both the short type and the long type, and a storage region characteristic of the archaeal FKBP-type PPIase in the FKBP domain. And two insertion sequences of the flap region (Maruyama T, Suzuki R, Furutani M., Front Biosci. (2004) 9: 1680-720). The long-type archaebacterium-derived FKBP-type PPIase has a C-terminal domain of about 100 to 150 amino acids on the C-terminal side of the FKBP domain having the above two insertion sequences. The FKBP-type PPIase of the present invention may be either a short type or a long type

[0028] In addition, the short type FKBP-type PPIase of the present invention has a part of a motif consisting of the amino acid sequence represented by SEQ ID NO: 1, 2 or 3 (hereinafter, referred to as "consensus sequence B"). It is preferable to have an amino acid sequence containing Xaa in the amino acid sequence represented by SEQ ID NO: 1, 2 or 3 is an arbitrary amino acid, which may be different from each other or the same amino acid. The consensus sequence B can be represented by the following general formula (I) in addition to SEQ ID NO: 1, 2 or 3.

 [Chemical 1]

Asp-A-Asn-His-B-Leu-Ala-Gly-X-Phe (I)

(Asp is aspartic acid, Asn is asparagine, His is histidine, Leu is mouth isine, Ala is alanine, Gly is glycine, A is any amino acid, B is any amino acid, X is 3 to 5 amino acids. Peptide consisting of any amino acid)

[0029] Furthermore, the short-type FKBP-type PPIase of the present invention has a motif consisting of the amino acid sequence represented by SEQ ID NO: 4 (hereinafter, referred to as "consensus sequence A"), and its N-terminal. Preferably, it is not included in the end region. Here, the N-terminal region refers to a region up to approximately 40 amino acid residues at the N-terminal region of the protein.

 The present inventors compared the amino acid sequences of nine short-type FK BP-type PPIases derived from mesophilic archae belonging to methanogens (estimated from the nucleotide sequence of the gene), and We found that it was classified into three groups depending on the presence or absence. That is, the short-type FKBP-type PPIase of Group 1 does not include the consensus sequence A in its N-terminal region, but does include the consensus sequence B in its amino acid sequence. The group 2 short-type FKBP-type PPIase does not include consensus sequence A in its N-terminal region, but includes an amino acid sequence in which consensus sequence B has Phe replaced with another amino acid in its amino acid sequence. In addition, the short type FKBP-type PP Iase of Group 3 has a consensus sequence A in its N-terminal region and a consensus sequence B in its amino acid sequence. FIG. 1 shows a diagram in which the amino acid sequences of the FKBP-type PPIases of Group 1, Group 2 and Group 3 are compared side by side. In FIG. 1, the amino acid sequence is represented by one letter notation.

 In FIG. 1, MaFKBP17.8 is a group 1 short-type FKBP-type PPIase derived from Methanosarcina acetivorans (SEQ ID NO: 8), and MmFKBP18.0 is a group 1 short-type FKBP-type PPIase derived from Methanos arcina mazei (SEQ ID NO: 6) MbFKBPl 9 is a group 1 short type FKBP type PPIase (SEQ ID NO: 10) derived from Mathanosarcina barkeri. MbFKBP21 is a group 3 short-type FKBP-type PPIase derived from M. barkeri (SEQ ID NO: 11), MmFKBPl7.9 is a group 3 short-type FKBP-type PPIase derived from M. mazei (SEQ ID NO: 12), and MaFKBP19 is an M This is a group 3 short type FKBP type PPIase (SEQ ID NO: 13) derived from Acetivorans. MaFKBP18.2 is M. acetivorans-derived group 2 short-type FKBP-type PPIase (SEQ ID NO: 14), and MmFKBP18.2 is M. mazei-derived group 2 short-type FKBP-type PPIase (SEQ ID NO: 15), MbFKBP18 Is a group 2 short-type FKBP-type PPIase (SEQ ID NO: 16) derived from M. barkeri.

[0032] Each consensus sequence will be described in more detail. First, the consensus sequence B is a sequence on the C-terminal side of each amino acid sequence shown in FIG. 1, “D * NH * LAG **** F” ( * Is an amino acid sequence corresponding to any amino acid). When this consensus sequence B is generalized, Asp—A—Asn—His—B—Leu—Ala—Gly—X—Phe (A is any amino acid, B is any amino acid, X is 3-5 arbitrary amino acids Amino acid sequence), which is the same as the amino acid sequence represented by SEQ ID NO: 1, 2 or 3. This consensus sequence B is a characteristic motif found in archaeal FKBP-type PPIase. The important amino acid in this consensus sequence B is Phe at the C-terminus, and as described above, short type FKBP-type PPIases can be classified into group 1 and group 2 depending on the type of amino acid at this position. Note that X in the consensus sequence B is a peptide having 3 to 5 arbitrary amino acid forces, but often has 4 amino acid forces as in the example of FIG.

 On the other hand, the consensus sequence A is an amino acid sequence corresponding to “GSGCJ, an N-terminal sequence of group 3 shown in FIG. 1. This amino acid sequence is represented by the amino acid sequence Gly— This consensus sequence A is a motif found in the region near the N-terminus of some FKBP-type PPIases derived from cold-tolerant archaea, and as described above, the short-type FKBP is determined by the presence or absence of this motif. The type PPIase can be classified into group 3 and other groups, that is, the short type FKBP type PPIase having this motif belongs to group 3. In the FKBP type PPIase of the present invention, the group having consensus sequence B 1 is particularly preferred, and it is preferable not to have consensus sequence B in the N-terminal region, even if the short-type FKBP-type PPIase of group 3 is used. By removing the consensus sequence A by making deleted modifying or deleting the N-terminal region, it can be converted into the group 1 Short FKBP-type PPIase.

[0034] Among these short-type FKBP-type PPIases, the FKBP-type PPIase of Group 1 has higher molecular chaperone activity than the FKBP-type PPIase of Group 2 or Group 3. Such FKBP-type PPIases of Group 1 include, for example, archaea belonging to the genus Methanosarcina such as M. mazei, M. acetivorans, and M. barkeri described above. Here, SEQ ID NO: 5 shows the amino acid sequence presumed to be the nucleotide sequence of the gene encoding the group 1 short type FKBP type P Plase derived from M. mazei, and SEQ ID NO: 6 shows only the amino acid sequence. Similarly, amino acid predicted to be the nucleotide sequence of the gene encoding group 1 short type FKBP type PPIase derived from M. acetivorans The acid sequence is shown in SEQ ID NO: 7, and the amino acid sequence alone is shown in SEQ ID NO: 8. Similarly, SEQ ID NO: 9 shows the amino acid sequence presumed to be the nucleotide sequence of the gene encoding the group 1 short type FKBP PPIase derived from M. barkeri, and SEQ ID NO: 10 shows only the amino acid sequence.

 On the other hand, among the FKBP-type PPIases of the present invention, as an example of the long type, the amino acid sequence estimated to be the nucleotide sequence of the gene encoding the long-type FKBP-type PPIase derived from Methanosarcin a acetivorans is shown in SEQ ID NO: 17, Only the amino acid sequence is shown in SEQ ID NO: 18.

 [0036] An FKBP-type PPIase having substantially the same amino acid sequence as the FKBP-type PPIase of the present invention and having substantially the same activity is also one of the present invention. Here, "having substantially the same amino acid sequence" means that one or more (preferably about 110, more preferably about 115) amino acids in the amino acid sequence are deleted. And one or more (preferably about 120, more preferably about 110, and more preferably about 115) amino acids added to the amino acid sequence, It means that one or more (preferably about 110, more preferably about 115) amino acids are substituted with another amino acid. Further, having substantially the same amino acid sequence means that the homology is at least 50% or more, preferably 70% or more, more preferably 80% or more, still more preferably 90% or more, and particularly preferably 95% or more. Means that. The homology of the amino acid sequence can be calculated using, for example, a commercially available multiple alignment program such as CulustalW.

 “Substantially the same activity” indicates that the molecular chaperone activity and the Z or PPIase activity are substantially the same. The molecular chaperone activity was evaluated by using, for example, rhodanese, quenate synthase, malate dehydrogenase, glucose phosphate dehydrogenase, etc. as model enzymes, and using this as a protein denaturant such as 6M guanidine hydrochloride. After denaturation

It can be performed by measuring the rate of denaturation protein regeneration and the rate of inhibition of denaturation protein aggregation that are initiated when the denaturant is diluted with a buffer containing the test substance (Kawata, Noo Science and Industrie). Riichi 56, 593—, 1998; Horowitz, Methods

Mol. Biol. 40, 361—, 1995; Taguchi, Biol. Chem. 269, 8529—, 19 94). The evaluation of PPIase activity can be performed by, for example, the chymotrypsin coupling method. Bacteriol. 1998, 180 (2): 388-394).

 [0038] The expression vector of the present invention is capable of expressing a fusion protein of the above-described FKBP-type PPIase of the present invention and a target protein. That is, the expression vector of the present invention has the first coding region encoding the FKBP-type PPIase of the present invention and at least one restriction enzyme site, and the second encoding region encoding the target protein in this restriction enzyme site. By inserting the region, a fusion protein of FKBP-type PPIase and the target protein can be expressed. Here, the fact that the fusion protein can be expressed means that the restriction enzyme site is located in the same reading frame as the first coding region. In other words, when the second coding region is inserted into the restriction enzyme site, the reading frames of the first coding region and the second coding region match, and the coding region of the first coding region and the second coding region are different. There is no intervening stop codon. The position of the restriction enzyme site may be upstream or downstream of the first coding region. When the restriction enzyme site is downstream of the first coding region, the first coding region needs to be effectively linked to the promoter. According to such an expression vector, the transcription of the first coding region is initiated by the promoter, and a fusion protein having FKBP-type PPIase at the N-terminal and a target protein at the C-terminal can be expressed.

 [0039] The above promoter may be appropriately selected according to the expression system to be used, and when expressed in a host, a promoter suitable for the host may be used. When the host is Escherichia coli, for example, Plac promoter, Ptac promoter, xylA promoter, AraB promoter, lambda promoter, T7 promoter and the like can be used. When expressed by a mammalian cell line, the genome of mammalian cells can be isolated, for example, a promoter such as the mouse metallothionein promoter, or a virus growing in these cells.Isolated, for example, baculovirus promoter, vaccinia virus 7. Promoters such as the 5K promoter can be used.

[0040] The expression vector of the present invention may further have a nucleotide sequence serving as a peptide linker between the first coding region and the restriction enzyme site. A representative example of such a peptide linker is a protease digested amino acid sequence. That is, the expression vector of the present invention In this case, it is preferable to have a nucleotide sequence encoding a protease-digested amino acid sequence between the first coding region and the restriction enzyme site. The nucleotide sequence encoding the protease digested amino acid sequence is transcribed and translated in the same reading frame as the first coding region and the second coding region, resulting in a protease digested amino acid sequence. That is, according to the expression vector of the present embodiment, it is possible to express a fusion protein in which FKBP-type PPIase and the target protein are linked via a protease-digested amino acid sequence. Then, the target protein can be cut out by allowing the corresponding protease to act on the fusion protein.

 [0041] The protease is not particularly limited and includes, for example, thrombin, factor Xa, precision protease, enterokinase and the like. As these proteases, for example, commercially available proteases can be used. Examples of the peptide linker other than the protease recognition amino acid sequence include a self-cleaving amino acid sequence of intin. That is, the target protein can be cut out from the fusion protein by utilizing the self-protein splicing function of Intin. The length of the nucleotide sequence encoding the peptide linker is not particularly limited, but it is preferably about 15 to 90 nucleotides, which preferably contains a large amount of the nucleotide sequence which is translated and becomes a neutral amino acid such as glycine-serine. Is preferred.

 [0042] The expression vector of the present invention may further contain another conventionally known base sequence.

 The other known nucleotide sequences are not particularly limited, and include, for example, a stability leader sequence for imparting the stability of the expression product, a signal sequence for imparting the secretion of the expression product, a neomycin resistance gene, a kanamycin resistance gene, and chloramphene. Marking sequences capable of conferring phenotypic selection in transformed hosts, such as the Nicol resistance gene, the ampicillin resistance gene, and the hygromycin resistance gene.

[0043] An expression vector in which the second coding region encoding the target protein has already been incorporated into the above-described restriction vector site of the expression vector is also one of the present invention. According to such an expression vector, an operation of incorporating the second coding region is unnecessary, and a fusion protein of FKBP-type PPIase and a target protein can be produced by performing transcription and translation as it is. The target protein encoded by the second coding region includes, for example, an antibody Or a partial fragment of an antibody. Examples of antibody partial fragments include Fab, Single chain Fv (scFv), Fc, and polypeptides in which two or more fragments of each domain of the antibody are linked by peptide bonds. In addition, the animal or subclass of the above antibody may be any. Thus, when a vector containing the second coding region encoding an antibody or a partial fragment of an antibody is used, a properly folded antibody is obtained without expression in an Escherichia coli host or the like. be able to. As a result, the antibody can be obtained more easily and in a larger amount than in the conventional case where it is not necessary to produce the antibody using an animal. The antibody-encoding gene can also prepare a hybridoma force, for example. And since the gene encoding the antibody obtained from a single hybridoma is single, the obtained antibody is a monoclonal antibody. On the other hand, in the case of obtaining polyclonal antibodies, for example, a plurality of different types of genes, each of which also obtains an antibody encoding an antibody, incorporates those genes into separate vectors, and prepares separate transformants. These transformants may be mixed and cultured.

 [0044] Other examples of the target protein encoded by the second coding region include an intracellular receptor protein. Most of intracellular receptor proteins that receive physiologically active substances are present in biological membranes. Since these intracellular receptor proteins selectively respond to various extracellular substances and transmit a variety of signals into the cells, clarification of their functions is of great interest as being directly linked to drug discovery. Have been. These intracellular receptor proteins form a structurally well-conserved family, which can be broadly classified into three types: endogenous ion channel, tyrosine kinase, and G protein-coupled. You.

[0045] In the ion channel endogenous type, when a ligand binds to a receptor, an ion channel existing in the receptor itself is opened, and Na +, Ca2 ^{+, and the} like are moved into the cell using an ion gradient inside and outside the cell. It is. The tyrosine kinase modulates the binding of the ligand to an increase in phosphorylation activity and amplifies the signal by triggering a series of cascades. In the G protein-coupled type, the receptor itself does not have an ion channel or enzymatic activity, and transmits information by binding of a ligand into cells via a G protein.

[0046] A number of drugs targeting intracellular receptor proteins have been developed, and many of them target protein-coupled receptors (GPCRs). Therefore, GPCR By identifying endogenous ligands and clarifying their functions and structures, it is expected that rapid drug development will be possible. The power of developing large-scale expression technology for GPCRs for crystallization and deuteration for ligand screening and structural analysis is essential.Expression of GPCRs has so far been considered impossible in E. coli and yeast. At present, various analyzes are being performed using trace samples expressed mainly in animal cultured cells such as CHO, COS-7, and HEK. However, according to the expression vector of the present invention, a recombinant protein can be produced inexpensively and in large quantities by using Escherichia coli or the like for the intracellular receptor protein.

[0047] The transformant of the present invention has the second coding region already incorporated in the above expression vector! /, Containing an expression vector. By culturing the transformant of the present invention, a fusion protein of FKBP-type PPIase and the target protein can be produced. The host from which the transformant of the present invention is derived is not particularly limited as long as it is compatible with the properties of the expression vector of the present invention.For example, prokaryotes such as bacteria, yeast, fungi, plants, insect cells, and mammals Cells and the like. Here, the expression that the host is compatible with the characteristics of the expression vector means that the expression vector is replicable in the host and that the first coding region and the second coding region can be transcribed and translated. In particular, in order for transcription to be performed normally, it is necessary to select a host in which the promoter on the expression vector works normally.

 [0048] Among the above-mentioned hosts, Escherichia coli is particularly suitable because it is easy to handle and has a rich host vector system. When Escherichia coli is used as a host, the fusion protein transcribed and translated from the expression vector may be expressed in the cytoplasm or in the periplasmic region. When expressing a fusion protein in the periplasmic region, an expression vector may be constructed so that a signal sequence is added to the 5 'end of the fusion protein, and introduced into E. coli.

 [0049] As a method for introducing the expression vector into the host when preparing the transformant of the present invention, various known methods can be used. For example, calcium phosphate precipitation, electroporation, liposome fusion, Nuclear injection, viral or phage infection and the like.

[0050] It should be noted that the expression vector of the present invention may be used in addition to a host for introduction into a host, such as a terrestrial or eukaryotic organism. Cell-free translation system using extracts and the like (Spirin, AS, 1991, Science, 11, 2656-2664: Falcone, D. et al., 1991, Mol. Cell. Biol., 11, 2656-2664) It can also be expressed. The cell-free translation system is effective when the target protein is toxic to the host.

[0051] The main embodiment of the fusion protein of the present invention is a fusion protein of an FKBP-type PPIase derived from a mesophilic archaea belonging to methanogen and a target protein. According to the fusion protein of the present invention, even when the target protein is expressed alone, the protein is not correctly folded and becomes an abnormal protein.By expressing the protein as a fusion protein with FKBP-type PPIase, the target protein was correctly folded. It can be expressed as a normal protein. As a result, the productivity of the normal target protein can be significantly improved. In addition, the target protein can also extract the fusion protein power by an appropriate method. For example, if a protease recognition amino acid sequence is inserted between the FKBP-type PPIase and the target protein, the target protein can be cut out and isolated by allowing the protease to act on the fusion protein. The fusion protein of the present invention can be produced, for example, by transcribing and translating the first coding region and the second coding region on the above-mentioned expression vector. Furthermore, if a nucleotide sequence encoding a protease recognition amino acid sequence is inserted between the first coding region and the second coding region, a fusion protein having a protease recognition amino acid sequence between the FKBP-type PPIase and the target protein can be produced. Can be built.

[0052] The method for producing a target protein of the present invention utilizes the action of a FKBP-type PPIase derived from a mesophilic archaeon belonging to methanogens to produce a correctly folded normal target protein. At this time, there are mainly two modes in which the FKBP-type PPIase acts on the target protein. One embodiment is a fusion expression method in which a target protein is expressed as a fusion protein with FKBP-type PPIase as described above. In the case of the fusion expression method, a step of cutting out the target protein is required. Another embodiment is a co-expression method in which FKBP-type PPIase and a target protein are independently expressed simultaneously. According to the co-expression method, it is possible to reduce the number of production steps without cutting out the target protein. In the case of a co-expression method using a host, the FKBP-type PPIase gene and the target protein The genes may be present on the same expression vector or incorporated into separate expression vectors. When they are present on the same expression vector, for example, each gene may be inserted downstream of a separate promoter. Furthermore, it is also possible to incorporate the FKBP-type PPIase gene into the host's genome and the target protein gene into an expression vector.

 [0053] The FKBP-type PPIase and the fusion protein of the present invention can be purified by a method generally used for protein purification, that is, by combining salting out, membrane separation, various types of chromatography, and the like. . In particular, by utilizing the fact that the molecular surface of the FKBP-type PPIase of the present invention is rich in hydrophobic amino acids, the FKBP-type PPIase and the fusion protein of the present invention can be purified with high purity by using hydrophobic interaction chromatography. can do. For example, FKBP-type PPIase or fusion protein is adsorbed to the resin via the hydrophobic group immobilized on the resin or the like, and the resin is washed under conditions such that the FKBP-type PPIase or the fusion protein is not eluted, and impurities are removed. Components can be removed. Examples of the hydrophobic group include an alkyl group of about C4 to C20 and a phenyl group. In addition, a sample containing FKBP-type PPIase and a fusion protein to be used for purification can be prepared, for example, from a culture of a transformant containing those genes.

 [0054] The FKBP-type PPIase and the fusion protein of the present invention can also be purified by utilizing the affinity between FKBP-type PPIase and FK506. That is, a carrier carrying FK506-rapamycin and its analogous conjugate is prepared, and a sample containing FKBP-type PPIase or fusion protein is brought into contact with the carrier, and FKBP-type PPIase or fusion protein is placed on the carrier. Can be captured. Then, the carrier force FKBP-type PPIase or fusion protein may be recovered by an appropriate method.

[0055] In addition, the histidine tag, which has a strength of about 6 histidine residues, can be easily expressed and introduced into the terminus of the FKBP-type PPIase or fusion protein of the present invention to facilitate the purification thereof. That is, the FKBP-type PPIase or fusion protein having a histidine tag at the end binds to a carrier chelated with a metal such as nickel via the histidine tag. Specifically, a sample containing an FKBP-type PPIase or a fusion protein is contacted with a carrier chelated with a metal such as nickel, and the FKBP is placed on the carrier via a histidine tag. Type PPIase or fusion protein can be captured. After that, it can be dissolved in imidazole or the like to recover the carrier strength of FKBP-type PPIase or fusion protein! Examples of a method using such a tag include a method in which daltathione S-transferase or a part thereof is used as a tag and purification by affinity chromatography using daltathione resin, and a method in which a maltose-binding protein or a part thereof is used as a tag and maltose conjugate is used as a tag. There is a method of purifying with fat. In addition, these tags may be introduced into the N-terminal side and the C-terminal side of the PPIase or the fusion protein, or may be introduced into both.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to only these Examples.

 Example 1

 In this example, an expression vector capable of expressing a fusion protein of a short-type FKBP-type PPIase (MmFKBP18.0) derived from an archaebacterium Methanosarcina mazei belonging to a methanogen and a target protein was constructed. MmFKBP18.0 is a short type FKBP type PPIase of group 1 type.

 [0058] A bacterial pellet was recovered from a bacterial suspension of Methanosarcina mazei Goel (DSM3647), and genomic DNA was recovered by phenol Z-cloth form treatment and ethanol precipitation. On the other hand, the Mml8-F1 primer shown in SEQ ID NO: 19 and the Mml8-R1 primer shown in SEQ ID NO: 20 were used as primers for PCR with reference to the base sequence of the known MmFKBP18.0 gene shown in SEQ ID NO: 5. Synthesized. Next, using the obtained genomic DNA as type III, PCR was performed using the Mml8-F1 primer and the Mml8-R1 primer as a primer pair to amplify the MmFKBP18.0 gene. When the base sequence of the amplified DNA fragment was confirmed, it was consistent with the base sequence of the known MmFKBP18.0 gene. At both ends of the amplified DNA fragment, an Ncol site derived from the 5 'end of Mml8-F1 and a Spel site derived from the 3' end of Mml8-Rl were introduced.

On the other hand, the linker Throm-F2 represented by SEQ ID NO: 39 and the linker Throm-R2 which is a complementary chain thereof were synthesized. The linker Throm-F2 has a Spel site on the 5 'side, an EcoRI site on the 3' side, and a BamHI site and an Ndel site for introducing the gene of the target protein therein. Furthermore, the target protein can be It has a nucleotide sequence that is translated into a thrombin-recognizing amino acid sequence upstream of the BamHI site so that it can be cut out by the enzyme (Figure 2). Next, the linker Throm-F2 and the linker Throm-R2 were annealed to prepare a double-stranded linker Throm.

[0060] The amplified DNA fragment containing the MmFKBP18.0 gene was treated with Ncol / Spel, and the double-stranded linker Thr was treated with SpelZEcoRI, respectively. The expression vector pMml8F2 was constructed by inserting the MmFKBP18.0 gene—double-stranded linker in the order of: The structure of the expression vector pM ml8F2 is shown in FIG. That is, the expression vector pMml8F2 has a MaFKBP18.0 gene serving as a first coding region, a base sequence encoding a thrombin-recognizing amino acid sequence, a BamHI site, and an Ndel site in sequence downstream of the T7 promoter. Then, a fusion protein of MmFKBP18.0 and the target protein can be expressed by inserting a gene encoding the target protein serving as the second coding region into the BamHI site or the Ndel site. The expressed fusion protein has a thrombin-digested amino acid sequence between MmFKBP18.0 and the target protein.

 Example 2

 In this example, an expression vector capable of expressing a fusion protein of a short-type FKBP-type PPIase (MbFKBP19.0) derived from the archaebacterium Methanosarcina barkeri belonging to methanogens and a target protein was constructed. MbFKBP19.0 is a group 1 type short type FKBP type PPIase.

[0062] A bacterial pellet was recovered from a bacterial suspension of Methanosarcina barkeri type strain (DSM800), and genomic DNA was recovered in the same manner as in Example 1. With reference to the base sequence of the known MbFKBP19.0 gene shown in SEQ ID NO: 9, the Mbl9-F1 primer shown in SEQ ID NO: 21 and the Mbl9-R1 primer shown in SEQ ID NO: 22 were synthesized as primers for PCR. . Next, using the obtained genomic DNA as type III, PCR was carried out using the Mbl9-F1 primer and the Mb19-R1 primer as a primer pair to amplify the MbFKBP19.0 gene. When the base sequence of the amplified DNA fragment was confirmed, it was consistent with the base sequence of the known MbFK BP19.0 gene. At both ends of the amplified DNA fragment, Sphl sites derived from the 5 'end of Mb 19-F1 and S The pel site was introduced. Next, in the same manner as in Example 1, an expression vector pMbl9F2 was constructed by inserting an amplified DNA fragment containing the MbFKBP19.0 gene and a double-stranded linker, Throm, into pET21 plasmid DNA. That is, the expression vector pMbl9F2 has the configuration of the expression vector pMml8F2 shown in FIG. 2, in which the MbFKBP19.0 gene replaces the MmFKBP18.0 gene.

 Example 3

 In this example, an expression vector capable of expressing a fusion protein of a short-type FKBP-type PPIase (MaFKBP17.8) derived from an archaebacterium Methanosarcina acetivorans belonging to a methanogen and a target protein was constructed. MaFKBP17.8 is a group 1 short-type FKBP-type PPIase.

 [0064] A bacterial pellet was recovered from a bacterial suspension of Methanosarcina acetivorans C2A (DSM2834), and genomic DNA was recovered in the same manner as in Example 1. Referring to the known base sequence of the MaFKBP17.8 gene shown in SEQ ID NO: 7, Mal7.8.-F1 primer shown in SEQ ID NO.23 and Mal7.8.R1 shown in SEQ ID NO.24 as primers for PCR Primers were synthesized. Next, PCR was carried out using the obtained genomic DNA as type III and Mal7.8-F1 primer and Mal7.8-R1 primer as a primer pair to amplify the MaFKBP17.8 gene. When the nucleotide sequence of the amplified DNA fragment was confirmed, it was consistent with the known nucleotide sequence of the MaFKBP17.8 gene. An Ncol site derived from the 5 'end of Mal7.8-F1 and a Spel site derived from the 3' end of Mal7.8-R1 were introduced into both ends of the amplified DNA fragment. Next, an expression vector pMal7F2 was constructed by inserting an amplified DNA fragment containing the MaFKBP17.8 gene and a double-stranded linker Throm into pET21 plasmid DNA in the same manner as in Example 1. That is, the expression vector pMal7F2 has a configuration in which the MaFKBP17.8 gene replaces the MmFKBP18.0 gene in the configuration of the expression vector pMml8F2 shown in FIG.

 Example 4

In this example, an expression vector capable of expressing a fusion protein of Methanosarcina acetivorans-derived long type FKBP-type PPIase (MaFKBP28.0) and a target protein was constructed. [0066] 1. Construction of expression vector

 With reference to the known nucleotide sequence of MaFKBP28.0 gene shown in SEQ ID NO: 17, Ma28-F1 primer shown in SEQ ID NO: 37 and Ma28-Rl primer shown in SEQ ID NO: 38 were synthesized as primers for PCR. . Next, the genomic DNA of Met hanosarcina acetivorans C2A (DSM2834) prepared in Example 3 was subjected to PCR using the Ma28-F1 primer and the Ma28-R1 primer as a primer pair to amplify the MaF KBP28.0 gene. did. When the nucleotide sequence of the amplified DNA fragment was confirmed, it was consistent with the known nucleotide sequence of the MaFKBP28.0 gene. An Ncol site derived from the 5 'end of Ma28-F1 and a Spel site derived from the 3' end of Ma28-R1 were introduced into both ends of the amplified DNA fragment. Next, the MaFKBP28.0 gene and the double-stranded linker Throm were inserted into the NcolZEcoRI site of the pET21d plasmid DNA in the same manner as in Example 1 to construct an expression vector pMa28F2. That is, the expression vector pMa28F2 has a configuration in which the MaFKBP28.0 gene is substituted for the MmFKBP18.0 gene in the configuration of the expression vector pMml8F2 shown in FIG.

 Example 5

 In this example, MaFKBP17.8 was produced and purified.

 [0068] 1. Production of MaFKBP17.8

 The expression vector pMal7F2 prepared in Example 3 was introduced into E. coli BL21 (DE3) strain (Novagen) to obtain a transformant. A 2 L Erlenmeyer flask was charged with 700 mL of 2 XYT medium (16 g / L yeast extract, 20 g / L bactotryptone, 5 g ZL NaCl, pH 7.5) containing lOO ^ g / mL ampicillin, and the resulting transformation was performed. The body was inoculated 2-3 times with a platinum loop and rotated at 35 ° C. for 24 hours (110 rpm). After completion of the culture, the culture was centrifuged (100,000 pm X 10 minutes) to collect the cells. The obtained cells were suspended in 20 mL of 25 mM HE PES buffer (pH 6.8) containing ImM EDTA and stored frozen at -20 ° C.

[0069] The cell suspension that had been cryopreserved was thawed and sonicated to disrupt the cells. The cell lysate was centrifuged to separate the supernatant (soluble fraction) and the sediment (sediment fraction). Further, the precipitate fraction was suspended in a 25 mM HEPES / lmM EDTA buffer (pH 6.8) containing 4% Triton X-100 and treated for 30 minutes to solubilize the membrane components. Centrifuge the suspension and settle. Was recovered. This operation was repeated twice, and the obtained precipitate was suspended in 20 mL of 25 mM HEPES / ImM EDTA buffer (pH 6.8) to prepare an inclusion body fraction. 10 g (5 L) of the soluble fraction and 5 L of the inclusion body fraction were subjected to SDS-PAGE, and the gel was stained with Coomassie brilliant blue (CBB). Figure 3 shows a photograph of the gel. In FIG. 3, the left lane is the soluble fraction, and the right lane is the precipitate fraction. That is, the band (arrow) corresponding to Ma FKBP17.8 was detected only in the soluble fraction. From the above, MaFKBP17.8 was recovered in a soluble state without becoming an inclusion body.

2. Purification of MaFKBP17.8

 The obtained soluble fraction was repeatedly subjected to hydrophobic interaction chromatography under the following conditions and column purification in the order of gel filtration, whereby MaFKBP17.8 was purified to almost a single unit. At the stage of hydrophobic interaction chromatography, MaFKBP17.8 was almost single.

 (a) Conditions for hydrophobic interaction chromatography

Column used: HighTrap Phenyl FF

 (Capacity 5 mL, manufactured by Amersham Biosciences)

Eluent

 Solution A: 10 mM phosphate buffer (pH 6.8) Z500 mM ammonium sulfate

 Solution B: 10 mM phosphate buffer (pH 6.8)

Elution conditions: 0-50 min: B-100 0% linear gradient elution

 50-70 minutes: 100% isocratic elution of solution B

Flow rate: 3mLZ min

Column temperature: room temperature

 (b) Gel filtration conditions

Column used: HiLoad 26/60 Superdex 200pg column

 (26 mm X 60 cm, manufactured by Amersham Bioscience)

Eluent: lOOmM sodium phosphate buffer (pH 7.0, containing 0.15M NaCl) Elution conditions: Isocratic elution Column temperature: room temperature

Next, the PPIase activity of the purified MaFKBP17.8 was evaluated by the following procedure. PPIase activity was measured according to the chymotrypsin coupling method. That is, in 2 mL of 10 OmM phosphate buffer (pH 7.8), 2.5-25 nM of MaFKBP17.8, N-sue-Ala-Leu-Pro-Phe-p-trophy, a substrate peptide of PPIase, -Lido (Peptide Institute) was cultivated to a final concentration of 2 / zM and pre-incubated at 15 ° C for 2 minutes. Next, chymotrypsin was added to a final concentration of 2 M, and the PPIase activity was evaluated by measuring the absorbance of the released troarlide. As a result, the PPIase activity (kcat / Km) of MaFKBP17.8 was 3.34s-M- ¹ . This value is based on the PPIase activity of FKBP-type short-type PPIase derived from thermophilic 'hyperthermophilic bacteria' described below, ie, 0.38 s " ¹ -μ

Hyperthermophilic archaea Thermococcus sp. KS- 1 PPIase (TcFKBP18 ) of 0. 35s- ¹ 'μ hyperthermophilic archaea Methanococcus jannaschii derived from PPIase of (MjFKBP18) 0. 92s- Μ- ¹ of (Ideno et al., Gene 2002 Jun 12; 292 (1-2): 57-63).

 Example 6

 In this example, MmFKBP18.0 was produced and purified.

 [0073] 1. Production of MmFKBP18.0

 The expression vector pMml8F2 prepared in Example 1 was introduced into E. coli BL21 (DE3) to obtain a transformant. Next, the transformant was cultured in the same manner as in Example 5, and after the cells were collected, the cell suspension was frozen and stored at 20 ° C. Further, the bacterial cell suspension was subjected to ultrasonic treatment in the same manner as in Example 5 to obtain a supernatant fraction (soluble fraction). When the supernatant fraction ΙΟ / zg was subjected to SDS-PAGE, a dark band corresponding to MmFKBP18.0 was detected, and it was confirmed that MmFKBPl8.0 was expressed in a large amount in the soluble fraction. .

 [0074] 2. Purification of MmFKBP18.0

The obtained supernatant fraction was subjected to hydrophobic interaction chromatography and gel filtration in the same manner as in Example 5, and anion exchange chromatography under the following conditions, and column purification was repeated.From this, MmFKBP18. 0 was purified to almost unity. (c) Anion exchange chromatography conditions

 Column used: DEAE Toyopearl column

 (16mm X 60cm; East USSR)

 Eluent

 Solution A: 25mM HEPES- KOH buffer (pH 6.8)

 Solution B: 25mM HEPES containing 0.5M NaCl—KOH buffer (pH 6.8) Elution conditions: 0-300 minutes: Linear gradient elution of solution B 0-100%

 300—420 minutes: Isocratic elution of 100% of solution B

 Flow rate: lmLZ min

 Column temperature: room temperature

 Example 7

 In this example, MbFKBP19.0 was produced and purified.

 1. Manufacturing of MbFKBP19.0

 The expression vector pMbl9F2 prepared in Example 2 was introduced into E. coli BL21 (DE3) to obtain a transformant. Next, the transformant was cultured in the same manner as in Example 5, and after the cells were collected, the cell suspension was frozen and stored at 20 ° C. Further, the bacterial cell suspension was subjected to ultrasonic treatment in the same manner as in Example 5 to obtain a supernatant fraction (soluble fraction). When 10 / z g of the supernatant fraction was subjected to SDS-PAGE, a dark band corresponding to MbFKBP19.0 was detected, and it was confirmed that MbFKBP19.0 was expressed in a large amount in the soluble fraction.

 [0077] 2. Purification of MbFKBP19.0

 The obtained supernatant fraction was subjected to hydrophobic interaction chromatography, gel filtration, and anion exchange chromatography in the same manner as in Example 6, and MbFKBP19.0 was substantially purified by repeating column purification. Purified to single.

 Example 8

 In this example, MaFKBP28.0 was manufactured.

[0079] In the same manner as in Example 5, production of a transformant containing the expression vector pMa28F2, culture, cell disruption, preparation of a soluble fraction and a precipitate fraction, and SDS-PAGE were performed. Figure 4 shows a photograph of the gel. In FIG. 4, the lane on the left is the soluble fraction, and the lane on the right is Is the precipitate fraction. That is, the band (arrow) corresponding to MaFKBP28.0 was detected only in the soluble fraction. As described above, MaFKBP28.0 was recovered in a soluble state without becoming an inclusion body.

 Example 9

 [0080] In this example, it was confirmed that MaFKBP17.8 had a molecular chaperone activity.

[0081] Rhodanese was dissolved in a 50 mM sodium phosphate buffer (pH 7.8) containing 6 M guanidine hydrochloride and 5 mM DTT to a final concentration of 63 M, treated at 25 ° C for 60 minutes, and treated with rhodanese. Denatured. The resulting denatured rhodanese solution was diluted 60-fold with a 50 mM sodium phosphate buffer (pH 7.8) containing 10 M MaFKBP17.8 and 1 OmM DTT, and treated for 45 minutes as it was. After the treatment, the activity of the regenerated rhodanese was evaluated according to the method of holopitch (Methods in Molecular Biology 40, 361-368 (1995)). The value of the regenerated rhodanese activity was expressed as a relative value with the activity of native rhodanese as 100%, and was defined as the molecular chaperone activity. As a control, rhodanese activity was measured in a solution not supplemented with MaFKBP17.8. Fig. 5 shows the results. That is, in the absence of MaFKB P17.8, only about 5% of rhodanese was regenerated, whereas in the presence of MaFK BP17.8, about 76% of rhodanese was regenerated. As described above, it was shown that the denatured rhodanese was properly folded and regenerated to normal rhodanese by the action of MaFKBP17.8.

 Example 10

 [0082] In this example, an expression vector capable of expressing a fusion protein consisting of MaFKBP17.8 and rhodanese mosquito derived from a hyperthermophilic bacterium (Aeropyrum pernix) was constructed. Further, a transformant containing the expression vector was prepared. Furthermore, the fusion protein was produced by culturing the transformant. Further, the fusion protein was purified from the culture using hydrophobic interaction chromatography. Furthermore, the purified fusion protein was cut out from rhodanese to obtain rhodanese as a normal protein.

 [0083] 1. Construction of expression vector and preparation of transformant

Genomic DNA was prepared in the same manner as in Example 1 using the bacterial pellet of Aeropyrum pernix (JCM9820; the same as DSM11879) obtained from RIKEN. on the other hand, With reference to the known nucleotide sequence of the rhodanese gene derived from A. pernix shown in SEQ ID NO: 40, an ApTS-F1 primer shown in SEQ ID NO: 25 and an ApTS-Rl primer shown in SEQ ID NO: 26 were used as primers for PCR. Synthesized. Next, the obtained genomic DNA was designated as type III, PCR was performed using the ApTS-F1 primer and the ApTS-R1 primer as a primer pair, and the rhodanese gene derived from A. pernix was amplified. When the nucleotide sequence of the amplified DNA fragment was confirmed, it was consistent with the known nucleotide sequence of the rhodanese gene derived from A. pernix. At both ends of the amplified DNA fragment, an Ndel site derived from the 5 'end of ApTS-F1 and a Hindlll site derived from the 3' end of ApTS-R1 were introduced. Next, the amplified DNA fragment containing the A. pernix-derived rhodanese gene was treated with Ndel / Hindlll, inserted into the NdelZHindlll site of the expression vector pMal7F2 constructed in Example 3, and MaFKBP17.8 and the A. pernix-derived rhodanese gene. An expression vector capable of expressing a fusion protein with was constructed. That is, the expression vector comprises, in order, downstream of the T7 promoter, the MaFKBP17.8 gene serving as the first coding region, the amino acid sequence encoding the thrombin recognition amino acid sequence, and the A. pernix serving as the second coding region. It has a gene encoding rhodanese. Therefore, according to the expression vector, a fusion protein of MaF KBP17.8 and rhodanese derived from A. pernix can be expressed. Also, the expressed fusion protein has a thrombin-digested amino acid sequence between MmFKBP17.8 and A. pernix-derived rhodanese. Next, the constructed expression vector was introduced into Escherichia coli BL21 (DE3) strain to prepare a transformant.

2. Production of fusion protein

The produced transformant was cultured and recovered in the same manner as in Example 5, and then a soluble fraction and an inclusion body fraction of the cell homogenate were obtained. The soluble fraction and the inclusion body fraction were subjected to SDS-PAGE in the same manner as in Example 5, and the gel was stained with CBB. Figure 6 (a) shows a photograph of the gel. That is, a band (arrow) corresponding to the fusion protein of MaFKBP17.8 and rhodanese derived from A. pernix (shown as “MaFKBP17.8_rho fusion protein” in the figure) was observed in the 1S soluble fraction. From the above, A. pernix-derived rhodanese was synthesized in a soluble state without becoming an inclusion body by using a fusion protein with MaFKBPl 7.8. [0085] 3. Purification of fusion protein

 By repeating column purification of the obtained soluble fraction under the same conditions as in Example 5 in the order of hydrophobic interaction chromatography and gel filtration, a fusion protein of MaFKBP17.8 and A. pernix-derived rhodanese was purified. .

 [0086] 4. Production of Rhodanese

 1 U of thrombin was added to 1 mg of the purified fusion protein and treated at 22 ° C. for 16 hours. The reaction solution was subjected to SDS-PAGE, and the gel was stained with CBB. Fig. 6 (b) shows a photograph of the gel. The left lane shows the sample before thrombin addition, and the right lane shows the sample after thrombin addition. In other words, only the fusion protein of MaFKBP17.8 and A. pernix-derived rhodanese was detected in the sample before the thrombin-supplemented rice cake, and the fusion protein was not detected in the sample after the thrombin-added treatment, corresponding to rhodanese. A band and a band corresponding to MaF KBP17.8 were detected. From the above, it was confirmed that the thrombin recognition site of the fusion protein of MaFKBP17.8 and rhodanese derived from A. pernix was cleaved, and that rhodanese was cut out.

 As a comparative example, A. pernix-derived rhodanese gene was expressed alone by the following procedure. The NdelZHindlll digestion product of the A. pernix-derived rhodanese gene prepared in Example 10 was previously treated with NdelZHindlll! And inserted into pET21a plasmid DNA to construct an expression vector that expresses A. pernix-derived rhodanese alone. . The obtained expression vector was introduced into E. coli BL21 (DE3) strain to prepare a transformant. The prepared transformant was cultured and recovered in the same manner as in Example 4, and the soluble fraction and the inclusion body fraction of the obtained cell lysate were subjected to SDS-PAGE, and the gel was stained with CBB. . A photograph of the gel is shown in FIG. 6 (c). The left lane is the soluble fraction and the right lane is the precipitate fraction. That is, A. pernix-derived rhodanese (arrow) was detected only in the inclusion body fraction, and hardly detected in the soluble fraction.

 From the above, A. pernix-derived rhodanese becomes an inclusion body when expressed alone in Escherichia coli, but when it is made into a fusion protein with MaFKBP17.8, it does not become an inclusion body but is synthesized into a soluble fraction. Powerful.

Example 11 [0088] In this example, an expression vector capable of expressing a fusion protein consisting of MaFKBP17.8 and a Fab antibody fragment was constructed. Further, a transformant containing the expression vector was prepared. Furthermore, the fusion protein was produced by culturing the transformant.

 [0089] 1. Construction of expression vector and preparation of transformant

 Anti-HEL Fab antibody fragment expression vector pEHELFab-l (Ideno, Appi. Env. Microbiol. 68, 464-, 2002) derived from mouse was treated with NdelZBpull02I, and the anti-HEL Fab antibody fragment was subjected to agarose gel electrophoresis. The gene fragment was purified. In this gene fragment, the gene encoding the heavy chain of the Fab antibody fragment and the gene encoding the light chain of the Fab antibody fragment are arranged side by side. Are performed separately, and the heavy chain and the light chain of the Fab antibody fragment are synthesized independently of each other, not as fusion proteins. Next, by inserting the gene fragment of the obtained anti-HEL Fab antibody fragment into the NdelZBpul102I site of the expression vector pMal7F2 constructed in Example 3, the MaFKBP17.8 and the heavy chain of the Fab antibody fragment were inserted. An expression vector capable of expressing the fusion protein was constructed. That is, the expression vector comprises, in order, downstream of the T7 promoter, the overlapping of the MaFKBP17.8 gene serving as the first coding region, the amino acid sequence coding for the thrombin recognition amino acid sequence, and the Fab antibody fragment serving as the second coding region. It has a gene encoding a chain. Therefore, according to the expression vector, a fusion protein of MaFKBP17.8 and the heavy chain of the Fab antibody fragment can be expressed. The expressed fusion protein also has a thrombin-digested amino acid sequence between MaFKBP17.8 and the heavy chain of the Fab antibody fragment. The light chain of the Fab antibody fragment is independently synthesized by the gene encoding the light chain of the Fab antibody fragment located downstream of the gene encoding the heavy chain of the Fab antibody fragment. Next, the constructed expression vector was introduced into Escherichia coli BL21 (DE3) strain to prepare a transformant.

 [0090] 2. Production of fusion protein

The produced transformant was cultured and recovered in the same manner as in Example 5, and then a soluble fraction and an inclusion body fraction of the cell homogenate were obtained. The soluble fraction and the inclusion body fraction were subjected to SDS-PAGE in the same manner as in Example 5, and the gel was stained with CBB. As a result, the soluble fraction A major band of the fusion protein of MaFKBP17.8 and Fab heavy chain was observed. As a result of measuring the band density of the fusion protein with a densitometer from the electrophoresis image of the soluble fraction stained with CBB, about 19% of the total soluble protein derived from Escherichia coli was the fusion protein, and the fusion protein was produced in large quantities. It had been. No band corresponding to the light chain portion of Fab was observed.

[0091] As a comparative example, the Fab antibody fragment gene was expressed alone by the following procedure. The NdelZBpul 1021 digestion product of the Fab antibody fragment gene prepared in Example 11 was inserted into pET21a plasmid DNA previously treated with Ndel / Bpul 1021, to construct an expression vector that expresses the Fab antibody fragment alone. The obtained expression vector was introduced into Escherichia coli BL21 (DE3) strain to prepare a transformant. The prepared transformant was cultured and recovered in the same manner as in Example 5, and the soluble fraction and the inclusion body fraction of the obtained lysate were subjected to SDS PAGE. As a result, the heavy chain of the Fab antibody fragment was detected only in the inclusion body fraction. From the above, the heavy chain of the Fab antibody fragment becomes an inclusion body when expressed alone in E. coli, but when it is made into a fusion protein with MaFKBP17.8, it does not become an inclusion body but is synthesized into a soluble fraction. Helped.

 Example 12

 [0092] In this example, an expression vector capable of expressing a fusion protein consisting of MaFKBP17.8 and a scFv antibody fragment was constructed. Further, a transformant containing the expression vector was prepared. Furthermore, the fusion protein was produced by culturing the transformant.

 [0093] 1. Construction of expression vector and preparation of transformant

 The SCF-F3 primer shown in SEQ ID NO: 27 and the SCF-R3 primer shown in SEQ ID NO: 28 were synthesized as primers for PCR. Next, plasmid pAALSC (Iba et al.)

1997, Gene 194, 35-) was used as type III, PCR was performed using SCF-F3 primer and SCF-R3 primer as a primer pair, and mouse anti--tritrizozyme (HEL) scFv antibody fragment (D1.3) gene Was amplified. The amplified gene was inserted into a pT7 blue vector (Novagen) by TA cloning, treated with Ndel / Notl, and inserted into the Ndel / Notl site of pMal7F2 constructed in Example 3 to obtain MaFKBP17.8 and scFv. antibody An expression vector capable of expressing a fusion protein with the fragment was constructed. That is, the expression vector comprises, in order, downstream of the T7 promoter, a MaFKB P17.8 gene serving as a first coding region, an amino acid sequence coding for a thrombin recognition amino acid sequence, and a scFv antibody fragment serving as a second coding region. Has a gene encoding Therefore, according to the expression vector, a fusion protein of MaFKBP17.8 and a scFv antibody fragment can be expressed. The expressed fusion protein also has a thrombin-digested amino acid sequence between MaFKBP17.8 and the scFv antibody fragment. Next, the constructed expression vector was introduced into Escherichia coli BL21 (DE3) strain to prepare a transformant.

 [0094] The produced transformant was cultured and recovered in the same manner as in Example 5, and then a soluble fraction and an inclusion body fraction of the cell lysate were obtained. The soluble fraction and the inclusion body fraction were subjected to SDS-PAGE in the same manner as in Example 5, and the gel was stained with CBB. As a result, a major band of a fusion protein of MaFKBP17.8 and a mouse-derived antl-HEL scFv antibody fragment was observed in the soluble fraction. From the electrophoresis image of the soluble fraction stained with CBB, the band density of the fusion protein was measured with a densitometer, and as a result, about 18% of the total soluble protein derived from Escherichia coli was the fusion protein, and the amount of the fusion protein was large. Had been manufactured.

 [0095] As a comparative example, a mouse-derived anti-HEL scFv antibody fragment (D1.3) was expressed alone by the following procedure. The mouse-derived anti-ditrilysozyme (HEL) sc Fv antibody fragment (D1.3) gene fragment prepared in Example 12 was roughly treated with the restriction enzyme NdelZBpul l02I, and inserted into pET21a plasmid DNA. An expression vector expressing the scFv antibody alone was prepared. The obtained expression vector was introduced into Escherichia coli BL21 (DE3) strain to prepare a transformant. The obtained transformant was cultured and recovered in the same manner as in Example 5, and the soluble fraction and the inclusion body fraction of the obtained cell lysate were subjected to SDS-PAGE, and the gel was stained with CBB. did. As a result, it was found that the mouse-derived anti-HEL scFv antibody hardly expressed in the soluble fraction, but was expressed as an aggregate in the inclusion body fraction. As described above, the mouse-derived anti-HEL scFv antibody fragment becomes an inclusion body when expressed alone in Escherichia coli, but when it is made into a fusion protein with MaFKBP17.8, it is synthesized into a soluble fraction instead of an inclusion body. That helped.

Example 13 [0096] In this example, an expression vector capable of expressing a fusion protein consisting of MaFKBP17.8 and a human-derived serotonin (HTla) receptor was constructed. Further, a transformant containing the expression vector was prepared. Furthermore, the fusion protein was produced by culturing the transformant.

 [0097] 1. Construction of expression vector and preparation of transformant

 Based on the known nucleotide sequence information of the HTla receptor gene (NCBI code: HSSERR51), an HTla-F1 primer shown in SEQ ID NO: 29 and an HTla-R1 primer shown in SEQ ID NO: 30 were synthesized as primers for PCR. Next, PCR was performed using the human placenta cDNA library (Takara Bio Inc.) as type III and HTla-Fl primer and HTla-Rl primer as a primer pair to amplify the HTla receptor gene. After inserting the amplified DNA fragment into the pT7 blue T vector, its nucleotide sequence was confirmed, and it was consistent with the registration information in the database. At both ends of the amplified DNA fragment, an Ndel site derived from the 5 'end of HTla F1 and a Notl site derived from the 3' end of HT1a-R1 were introduced. Next, the amplified DNA fragment containing the HTla receptor gene was treated with NdelZNotl, inserted into the NdelZNotl site of the expression vector pMal7F2 constructed in Example 3, and expressed a fusion protein of MaFKBP17.8 and the HTla receptor. An expression vector was constructed. That is, the expression vector encodes, in order, downstream of the T7 promoter, a MaFKBP17.8 gene serving as a first coding region, an amino acid sequence coding for a thrombin-recognizing amino acid sequence, and an HTla receptor serving as a second coding region. It has a gene. Therefore, according to the expression vector, a fusion protein of MaFKBP17.8 and HTla receptor 1 can be expressed. The expressed fusion protein also has a thrombin-digested amino acid sequence between MaFKBP17.8 and the HTla receptor. Next, the constructed expression vector was introduced into E. coli BL21 (DE3) strain to prepare a transformant.

 [0098] 2. Production of fusion protein

The produced transformant was cultured and recovered in the same manner as in Example 5, and then a soluble fraction and an inclusion body fraction of the cell homogenate were obtained. The soluble fraction and the inclusion body fraction were subjected to SDS-PAGE in the same manner as in Example 5. The fusion protein is detected by gel staining with CBB, Both Western blotting using an anti-serotonin receptor antibody was performed. As a result, a fusion protein of MaFKBP17.8 and HTla receptor was detected only in the soluble fraction. As a result of measuring the band density of the fusion protein with a densitometer from an electrophoresis image of the soluble fraction stained with CBB, about 2% of the total soluble protein derived from Escherichia coli was the fusion protein.

 Example 14

 [0099] In this example, MaFKBP17.8 and a scFv antibody fragment were co-expressed in a host cell to produce a scFv antibody fragment.

 [0100] In the same manner as in Example 12, the gene of the mouse-derived anti-HEL scFv antibody fragment was amplified. This amplified DNA fragment was inserted into a pT7 blue T vector, treated with NdelZNotl, and previously treated with Ndel / Notl !, and inserted into pET21a plasmid DNA to construct an expression vector pETscFv of the above scFv. Next, pETscFv was used as a 铸 type, and PCR was performed using the T7-F1 primer shown in SEQ ID NO: 35 and the T7-R1 primer shown in SEQ ID NO: 36 as a primer pair, and the scFv containing the above scFv gene and T7 promoter portion Expression unit DNA fragment was amplified. At both ends of the amplified DNA fragment, a Sph I site derived from the 5 'end of the T7-F1 primer and a BamHI site derived from the 3' end of the T7-R1 primer were introduced. This amplified DNA fragment was inserted into the pT7 blue vector by TA cloning, treated with the restriction enzyme SphlZBamHI, and inserted into the SphlZBamHI site of pACYC184 plasmid (Wako Pure Chemical) to construct the co-expression vector pACscFv. did.

[0101] Two types of vectors, the coexpression vector pACscFv and the expression vector pMal7F2 prepared in Example 3, were introduced into the competent cell Escherichia coli JM109 (DE3) strain, and 100 μgZmL ampicillin and 100 μgZmL chloramphene were introduced. -Cultivated on LB agar medium containing Cole. The obtained colony was inoculated into 700 mL of 2XYT medium containing 100 μg / mL ampicillin and 100 μg ZmL chloramphenecol. After rotating the culture at 35 ° C (110 rpm), when the OD600 reached 1.6, add 7 mL of lOOmM IPTG, lower the culture temperature to 20 ° C, and culture for another 18 hours. Expression of anti-HEL scFv antibody fragments derived from 8 and mouse was induced. After culturing, centrifuge (10,000 rpm x 10 minutes) )). The collected cells were suspended in 20 mL of 25 mM HEPES buffer (pH 6.8) containing 1 mM EDTA, and stored frozen at -20 ° C. The cells were thawed, sonicated, and separated into a supernatant (soluble fraction) and a precipitated fraction, each of which was subjected to SDS-PAGE and stained with CBB. On the other hand, as a comparative example, the same operation was performed on recombinant E. coli into which only pACscFv had been introduced. As a result, in the recombinant E. coli in which only pACscFv was introduced, all the scFv antibody fragments were detected in the precipitate fraction, but in the recombinant E. coli in which both pACscFv and pMal7F2 were introduced, the scFv antibody fragment was found in the supernatant fraction. was detected. As described above, co-expression with MaFKBP17.8 enabled scFv to be expressed in the soluble fraction.

 Example 15

 [0102] In this example, an expression vector capable of expressing a fusion protein comprising MaFKBP28.0 and a scFv antibody fragment was constructed. Further, a transformant containing the expression vector was prepared. Furthermore, the fusion protein was produced by culturing the transformant.

 [0103] 1. Construction of expression vector

 The amplified DNA fragment containing the scFv antibody fragment gene prepared in Example 12 was treated with NdelZNotl, and inserted into the Ndel / Notl site of the expression vector pMa28F2 constructed in Example 4, where MaFKBP28.0 and the scFv antibody fragment were combined. An expression vector Ma28F2-scFv capable of expressing the fusion protein was constructed. That is, the expression vector Ma28F2-scFv comprises, in order, downstream of the T7 promoter, a MaFKBP28.0 gene serving as a first coding region, an amino acid sequence encoding a thrombin recognition amino acid sequence, and a scFv antibody fragment serving as a second coding region. It has an encoding gene. Therefore, according to the expression vector, a fusion protein of MaFKB P28.0 and a scFv antibody fragment can be expressed. The expressed fusion protein also has a thrombin-digested amino acid sequence between MaFKBP28.0 and the scFv antibody fragment. Next, the constructed expression vector Ma28F2-scFv was introduced into Escherichia coli BL21 (DE 3) to prepare a transformant.

 [0104] 2. Production of fusion protein

The prepared transformant was cultured and recovered in the same manner as in Example 5, and then disrupted. A liquid soluble fraction and an inclusion body fraction were obtained. The soluble fraction and the inclusion body fraction were subjected to SDS-PAGE in the same manner as in Example 5, and the gel was stained with CBB. Fig. 7 (a) shows a photograph of the gel. That is, a band (arrow) corresponding to a fusion protein of MaFKBP28.0 and an scFv antibody fragment (indicated as “MaFKBP28.O-scFv fusion protein” in the figure) was observed in the soluble fraction. As described above, the scFv antibody fragment was synthesized in a soluble state without becoming an inclusion body by using a fusion protein with MaFKBP28.0.

 Example 16

 [0105] In this example, an expression vector capable of expressing a fusion protein consisting of MaFKBP28.0 and Rhodanese derived from A. pernix was constructed. Further, a transformant containing the expression vector was prepared. Furthermore, the fusion protein was produced by culturing the transformant.

 [0106] 1. Construction of expression vector and preparation of transformant

 The amplified DNA fragment containing the A. pernix-derived rhodanese gene prepared in Example 10 was treated with NdelZHindlll, inserted into the NdelZ Hindlll site of the expression vector pMa28F2 constructed in Example 4, and MaFKBP28.0 and A. pernix-derived rhodanese. An expression vector pMa28F2-rho capable of expressing a fusion protein with E. coli was constructed. That is, the expression vector pMa28F2-rho is, in order, downstream of the T7 promoter, a MaFKBP28.0 gene serving as a first coding region, an amino acid sequence encoding a thrombin recognition amino acid sequence, and an A. pernix-derived rhodanese gene serving as a second coding region. Has a gene encoding Therefore, according to the expression vector, a fusion protein of MaFKBP28.0 and A. pernix-derived rhodanese can be expressed. The expressed fusion protein also has a thrombin digested amino acid sequence between MaFKBP28.0 and A. pernix-derived rhodanese. Next, the constructed expression vector pMa28F2-rho was introduced into Escherichia coli BL21 (DE3) to prepare a transformant.

 [0107] 2. Production of fusion protein

The produced transformant was cultured and recovered in the same manner as in Example 5, and then a soluble fraction and an inclusion body fraction of the cell homogenate were obtained. The soluble fraction and the inclusion body fraction were subjected to SDS-PAGE in the same manner as in Example 5, and the gel was stained with CBB. Figure 7 (b) shows a photograph of the gel. Show. That is, a band (arrow) corresponding to a fusion protein of MaFKBP28.0 and rhodanese derived from A. pernix (indicated as “MaFKBP28.O_rho fusion protein” in the figure) was observed in the soluble fraction. From the above, A. pernix-derived rhodanese was synthesized in a soluble state without becoming an inclusion body by making it a fusion protein with MaFKBP28.0. Example 17

 In this example, an expression vector capable of expressing a fusion protein capable of expressing MaFKBP28.0 and natural jellyfish-derived natural GFP (Green fluorescent protein) was constructed. Further, a transformant containing the expression vector was prepared. Furthermore, the fusion protein was produced by culturing the transformant.

 [0109] 1. Construction of expression vector and preparation of transformant

The GFP-F1 primer shown in SEQ ID NO: 31 and the GFP-R1 primer shown in SEQ ID NO: 32 were synthesized as primers for PCR. Next, the control vector GFP (contained in the Cell-free protein translation system kit manufactured by Roche Diagnostics, Inc.), which is an expression vector containing the native GFP gene derived from O jellyfish, was designated as 铸 type, and GFP-F1 PCR was performed using the primer and the GFP-R1 primer as a primer pair to amplify the native GFP gene derived from Oan jellyfish. A BamHI site derived from the 5 'end of GFP-F1 and an EcoRI site derived from the 3' end of GFP-R1 were introduced into both ends of the amplified DNA fragment. The amplified DNA fragment was inserted into the pT7 blue vector by TA cloning, and the nucleotide sequence was confirmed. As a result, the nucleotide sequence was identical to the nucleotide sequence of the wild type GFP gene derived from Jellyfish jellyfish registered in the database. After treating the amplified DNA fragment with BamHlZEcoRI, it is inserted into the site with BamHI / EcoRI of pMa28F2 constructed in Example 4, and an expression vector capable of expressing a fusion protein of MaFKBP28.0 and native jellyfish-derived GFP. Was built. That is, the expression vector encodes, in order, downstream of the T7 promoter, a MaFKBP28.0 gene serving as a first coding region, an amino acid sequence encoding a thrombin-recognizing amino acid sequence, and a native jellyfish-derived GFP serving as a second coding region. The gene has Therefore, according to the expression vector, it is possible to express a fusion protein of MaFKBP28.0 and native jellyfish-derived GFP. The expressed fusion protein was MaFKBP28.0 and It has a thrombin-digested amino acid sequence between native jellyfish-derived GFP. Next, the constructed expression vector was introduced into E. coli BL21 (DE3) strain to prepare a transformant.

 [0110] 2. Production of fusion protein

 The resulting transformant was cultured and collected in the same manner as in Example 5 (however, the culture temperature was 15 ° C.), and then a soluble fraction and an inclusion body fraction of the cell homogenate were obtained. The soluble fraction and the inclusion body fraction were subjected to SDS-PAGE in the same manner as in Example 5, and the gel was stained with CBB. A photograph of the gel is shown in FIG. 7 (c). In other words, a band (arrow) corresponding to a fusion protein of MaFKBP28.0 and natural GFP derived from Oan jellyfish (indicated as “MaFKBP28.0-GFP fusion protein” in the figure) was observed in the soluble fraction. From the above, natural type GFP derived from O jellyfish was synthesized in a soluble state without becoming an inclusion body by using a fusion protein with MaFKBP28.0. From the electrophoresis image of the soluble fraction stained with CBB, the band density of the fused protein was measured with a densitometer.As a result, about 28% of the total soluble protein derived from Escherichia coli was the fused protein and was expressed in large amounts. .

[0111] As a comparative example, the same operation was performed using a hyperthermophilic archaeon-derived FKBP-type PPIase. That is, the expression plasmid pEFEl—3 (Iida ゝ Gene 222, 249—, 1998) of the short-type FKBP-type PPIase (TcFKBP18) derived from the hyperthermophilic archaeon Thermococcus sp. PCR was performed using the TcFu-Fl primer shown and the TcFu-R2 primer shown in SEQ ID NO: 34 as a primer pair, and a DNA fragment containing the TcFKBP18 gene was amplified. An Ncol site derived from the 5 'end of TcFu-F1 and a Spel site derived from the 3' end of TcFu-R2 were introduced into both ends of the amplified DNA fragment. When the amplified DNA fragment was inserted into the pT7 blue T vector and the nucleotide sequence was confirmed, it was consistent with the nucleotide sequence registered in the database. After treating the amplified DNA fragment with the restriction enzyme NcolZSpel, it is inserted into the site of NcolZSpel of pMa17F2 constructed in Example 3 to obtain an expression vector capable of expressing a fusion protein of TcFKBP18 and native GFP derived from O jellyfish. It was constructed. That is, in this expression vector, the MaFKBP28.0 gene of the expression vector constructed in Example 17 was replaced with the TcFKBP18 gene. Using this expression vector, a transformant was prepared, cultured, and SDS-PAGE of the disrupted cells was carried out in the same manner as in Example 17. As a result, similar to Example 17 Then, a fusion protein of TcFKBP18 and native GFP derived from Oan jellyfish was detected in the soluble fraction. As a result of measuring the band density of the fusion protein with a densitometer from an electrophoresis image of the soluble fraction stained with CBB, about 19% of the total soluble protein derived from Escherichia coli was the fusion protein and was expressed in large amounts.

 [0112] A solution sample was prepared with the same concentration of each fusion protein of Example 17 and the comparative example, and irradiated with UVB. As a result, the fluorescence intensity of the sample containing the fusion protein with MaFKBP28.0 was about twice the fluorescence intensity of the sample containing the fusion protein with TcFKBP18. As described above, in the fusion protein between the FKBP-type P Plase obtained and the soluble jellyfish-derived natural GFP, the fusion protein with MaFKBP28.0 was more soluble than the fusion protein with TcFKBP18. The jellyfish-derived native GFP was correctly folded.

 [0113] The following table summarizes the nucleotide sequences, restriction enzyme sites, and SEQ ID NOs of the PCR primers used in the above Examples.

[table 1]

Restriction enzyme Primer Nucleotide sequence SEQ ID No. Site ml8-F1 5 '-GGCCATGGGATTGATTGTTATGACTGAG-3' Nco \ 19

Hm18-R1 5 '-CCACTAGTTCATTCCTCCACTG-3' Spe \ 20

Mb19-F1 5 "-GGGCATGCGATTCTCGTTTGTTTTATTGATTTTTATG-3 'Sph \ 21

Mb19-R1 5 '-CCACTAGTTCACTCCTCCAC-3' Spe \ 22

Ma17.8-F1 5 '-GGCCATGGGAACTGAAGAGACAATTAAAAAC-3, Nco \ 23

Ha17.8-R1 5 '-CCACTAGTTTATGCCTCCAATG-3' Spe \ 24

ApTS-F1 5 '-GGCATATGTCGAGGC 丌 GGCGTAGA- 3' Nde \ 25

ApTS-R1 5'-CTGAATTCTCAGGGTTCGTCCCCCTTCT-3 '/ "dil l 26

SCF-F3 5 '-ATCATATGAAATACCTATTGCCTACG-3' Nde \ 27

SCF-R3 5 '-ATGCGGCCGCCTATTACTCCAGCTTGGTCCCTC-3' Not \ 28

HT1a-F1 5 '-ATCATATGGATGTGCTCAGCC-3' Nde \ 29

HT1a-R1 5 '-ATGCGGCCGCCTAGCCGCCAG-3' Not \ 30

GFP-F1 5 '-GGGGATCCAGTAAAGGAGAAGAACTTTTCACT-3' BamW \ 31

GFP-R1 5 '-CCGAATTCTTATTTGTATAGTTCATCCATGCCATG-3' EcoRi 32

Tcfu- F1 5 '-GGCCATGGGAAAAGTTGAAGCTGGTGAT-3' Nco \ 33

Tcfu-R2 5 '-CCACTAGTAGCTTCTGAGTCCTCTTC-3' Spe \ 34

T7-F1 5 '-GCATGCTAATACGACTCACTATA-3' Sph \ 35

T7-R1 5 '-GGATCCCAAAAAACCCCTC-3' BamW \ 36

Ha28-F1 5 '-ACCCATGGGTGCAATTCAGAAAGGCGATTT-3' Nco \ 37

Ma28-R1 5 '-GGACTAGTTTCATGGGATTCTGAAGTCTCTTC-3 * Spe \ 38

* Underlines indicate restriction enzyme sites.

Claims

The scope of the claims

 [I] FKBP-type PPIase derived from mesophilic archaea belonging to methanogens

[2] The mesophilic archaeon is a Methanosarcina archaea.

The FKBP-type PPIase according to 1.

[3] The FKBP-type PPIase according to claim 1 or 2, which is a short type FKBP-type PPIase.

 [4] The FKBP-type PPIase according to any one of [1] to [3], wherein the FKBP-type PPIase has an amino acid sequence that partially includes the motif capable of amino acid sequence represented by SEQ ID NO: 1, 2 or 3.

 [5] The FKBP-type PPIase according to any one of claims 1, 2, 3 or 4, wherein the motif comprises the amino acid sequence represented by SEQ ID NO: 4;

[6] An FKBP-type PPIase having the amino acid sequence set forth in SEQ ID NO: 6.

[7] An FKBP-type PPIase having the amino acid sequence of SEQ ID NO: 8.

[8] An FKBP-type PPIase having the amino acid sequence set forth in SEQ ID NO: 10.

[9] The FKBP-type PPIase according to claim 1 or 2, which is a long-type FKBP-type PPIase.

 [10] An FKBP-type PPIase having the amino acid sequence set forth in SEQ ID NO: 18.

 [II] has substantially the same amino acid sequence as the FKBP-type PPIase according to claim 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, and has substantially the same activity. FKBP-type PPIase characterized by having.

 [12] a first coding region encoding the FKBP-type PPIase according to claim 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11, and at least one restriction enzyme site, An expression vector, which is capable of expressing a fusion protein of FKBP-type PPIase and a target protein by inserting a second coding region coding for the target protein into the restriction enzyme site.

 [13] The expression vector according to claim 12, wherein the restriction enzyme site is located downstream of the first coding region.

[14] A salt encoding a peptide linker is located between the first coding region and the restriction enzyme site. 14. The expression vector according to claim 12, which has a base sequence.

[15] The expression vector according to claim 14, wherein the peptide linker comprises a protease-digested amino acid sequence.

[16] An expression vector, wherein a second coding region encoding a target protein is incorporated into a restriction enzyme site of the expression vector according to claim 12, 13, 14, or 15.

17. The expression vector according to claim 16, wherein the second coding region is a gene encoding an antibody or a partial fragment of the antibody.

18. The expression vector according to claim 16, wherein the second coding region is a gene encoding an intracellular receptor protein.

[19] A transformant comprising the expression vector according to claim 16, 17 or 18.

[20] The transformant according to claim 19, wherein the host is Escherichia coli.

 [21] A fusion protein of the FKBP-type PPIase according to claim 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 and a target protein.

22. The fusion protein according to claim 21, wherein the fusion protein has a protease-digested amino acid sequence between the FKBP-type PPIase and the target protein.

[23] A fusion protein characterized in that the first and second coding regions incorporated in the expression vector according to claim 16 are transcribed and translated to synthesize a fusion protein of FKBP-type PPIase and a target protein. Production method.

[24] The first and second coding regions incorporated into the expression vector according to claim 16 are transcribed and translated to synthesize a fusion protein of FKBP-type PPIase and the target protein.

Then, a method for producing a target protein, comprising cutting out the target protein from the fusion protein.

[25] The expression vector has a base sequence encoding a protease-digested amino acid sequence between the first coding region and the second coding region, and comprises a first coding region and a base sequence encoding a protease-digested amino acid sequence. Transcribes and translates the second coding region, synthesizes a fusion protein having a protease-digested amino acid sequence between the FKBP-type PPIase and the target protein, and allows the protease to act on the fusion protein to convert the target protein. 25. The method for producing a target protein according to claim 24, wherein the target protein is cut out.

[26] The gene encoding the FKBP-type PPIase according to claim 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11, and the gene encoding the target protein are co-expressed in the same host. A method for producing a target protein, which comprises causing FKBP-type PPIase to act on the target protein in the host, solubilizing the target protein, and collecting the target protein from the soluble fraction of the host.

 [27] A sample containing the FKBP-type PPIase according to claim 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 is subjected to hydrophobic interaction chromatography to obtain the FKBP-type PPIase. A method for purifying FKBP-type PPIase, comprising removing components other than the above.

 [28] A sample containing a fusion protein of the FKBP-type PPIase according to claim 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11, and a target protein is subjected to hydrophobic interaction chromatography. A method for purifying a fusion protein, comprising removing components other than the fusion protein by subjecting the fusion protein to chromatography.