Peptidyl prolyl-cis.trans-isomerase

Abstract: An E . coli PPIase-β characterized by possessing the following properties: (1) acting on and isomerizing the bond X aa -Pro (wherein X aa stands for any amino acid and Pro for L-proline), (2) exhibiting a single molecular weight of about 20,000 daltons in the sodium dodecyl sulfate-polyacrylamide concentration gradient gel electrophoresis, (3) exhibiting a single isoelectric point of about 5.0 in the isoelectric focusing, and (4) not being inhibited by CsA.

Description: The present invention relates to an Escherichia coli peptidyl prolyl-cis·trans isomerase-β and genes coding for the same. Peptidyl prolyl-cis.trans isomerase (PPIase) accelerates protein folding reaction by catalyzing the isomerization of prolyl peptide bonds in the protein. It is useful as means for activating an inactive protein produced by genetic engineering and as a reagent for enzymological analysis. Its corresponding gene coding is useful for the production of the enzyme by genetic engineering, and is also expected to be used as starting material for the gene which allows for the production by genetic engineering of the enzyme derivatives in which the enzymatic activity, intracellular localizability, substrate specificity and stability thereof have been altered. The present application further describes means for efficient production of a useful protein with a correct conformation by expressing the above-mentioned gene in the same cells as that in which other gene coding for the useful protein is expressed. PPIase was found in the porcine kidneys [Fisher, G., Bang, H. & Mech, C. Biomed. Biochim. Acta , 43 , 1101-1111 (1984)], is known to have the enzymatic activity of isomerizing X aa -Pro bond (wherein Xaa stands for any amino acid and Pro for L-proline) [Fisher, G., Bang, H & Mech, C. Biomed . Biochim . Acta , 43 , 1101-1111 (1984)] and accelerate protein folding reaction of some denatured proteins such as immunoglobulin L chain and ribonuclease T 1 [Lang, K., Schmid, F. & Fisher, G, Nature 329 , 268-270 (1987)]. The amino acid sequence of the enzyme purified from porcine kidneys was shown to be identical with that of Cyclophilin, a protein known to bind to an immunosuppressive agent, cyclosporine A (CsA) which has found to inhibit the PPIase activity. Since the immunosuppressive activity is directly proportional to the binding ability of CsA derivatives to cyclophilin, which represents the most of the CsA-binding activity in lymphoid cells, it is inferred that the action of CsA, for example, immunosuppressing action in T-cells, is mediated through the inhibition of the PPIase activity [Takahashi, N., Hayano, E. & Suzuki, M. Nature 337 , 473-475 (1989)]. The CsA binding and the PPIase activities were found in almost all organs and in nearly all species. However it is not known how CsA acts specifically on immune system, particularly on T cells, during an organ transplantation, has not been explained. Rhodopsin, a visual pigment occurring in animal retinas, consists of a chromophore, 11-Cis-retinal, bonded to opsin in the protein portion of rhodopsin, and is associated with visual transduction in the photoreceptor cells in which the chromophore is gradually converted and the maximum wavelength of absorption is consequently varied under the influence of light. In Drosophila a mutant whereby the conversion of the precursor opsin to the rhodopsin is inhibited, is known. The nina A gene responsible for this inhibition has been isolated and nucleotide sequence thereof has been determined, and as a result, it has been demonstrated that the nina A gene codes for a protein showing a homology to cyclophilin in the amino acid sequence. Accordingly, since the nina A codes for a cyclophilin-like protein and the cyclophilin is identical with the PPIase, the nina A gene probably encodes a protein possessing a PPIase activity, thus its activity is assumed to effect the formation of rhodopsin from its precursor opsin by controlling its folding reactions. The nina A gene is exclusively expressed in the head part containing photoreceptor cells, and in the other parts of body, gene fragments hybridizing with the nina A gene and possessing a different size therefrom are detected. Consequently, it is inferred that the nina A gene is specifically expressed in the photoreceptor cells and functions only in the formation of rhodopsin [Shieh, B.-H., Stamnes, M.A Seavello, S., Harris, G.L. & Zuker, C.S., Nature, 338, 67-70 (1989); and Schneuwly, S., Shortridge, R.D., Larrivee, D.C. Ono, T., Ozaki, M. & Pak, W.L. Proc. Natl. Acad. Sci USA. , 86 (1989)]. Likewise, if a PPIase is specifically expressed in T cells the presence of the T-cell specific form may offer an explanation for the specific effect of CsA on the T cells. This assumption is supported by the observation that many gene copies capable of hybridizing with the cyclophilin gene are present in mammalian cells [Haendler, B., Hofer Warbinek, R. & Hofer, E., EMBO J , 6 , 947-950 (1987)]. Only one kind of cyclophilin is confirmed to be expressed in each cell so far analyzed, and no multiplicity is found in the protein so expressed. A proof of the presence of several cyclophilins or PPIases in one species may well be theoretically accepted as evidence that each PPIase has a specific protein substrate. In the case of Neurospora the presence of two species of cyclophilin mRNA transcribed from a single gene is known. One of these mRNAs codes for the cyclophilin molecule present in the cytoplasm, and another mRNA codes for a mitochondrial form having an additional signal sequence for its translocation to mitochondria. The latter protein, after translocation in the mitochondrion, is processed and form a molecular species identical with the cyclophilin present in the cytoplasm. In this case, therefore, one molecular species of active protein is actually present and the multiplicity is ascribable to difference in the localization of one protein in the cell [Tropschug, M., Nicholson, D.W. Hartl, F.-U., Köhler, H. Pfanner, N., Wachter, E. & Neupert, W. J . Biol. Chem. 263 , 14433-14440 (1988)]. The catalytic effect of PPIase on the protein folding was investigated in nine kinds of proteins, i.e., concanavalin A, prothrombin, ribonuclease A, ribonuclease T 1 , cytochrome C, β-lactoglobulin, meiokinase, chymotrypsinogen, and pepsinogen, which are known to be such that, during the refolding of the protein from the denatured state to the active state, the isomerization of the prolyl-containing peptide bonds constitutes the rate-determining step in the reconstruction of the denatured protein. However the refolding of only two kinds of these proteins, i.e., ribonuclease T and cytochrome C were found to be accelerated by PPIase [Lin, L-N., Hasumi, H. & Brands, J.F. Biochim. Botphys, Acta 956, 256-266 (1988)]. These results suggest that one species of PPIase can act upon limited species of protein substrates. Although, based on the facts described above, the presence of multiple form of PPIase acting on restricted kinds of proteins is presumed, the presence thereof has not been actually proved. The theory that multiple forms of PPIase are present in one species of organism, and that the substrate specificity of a given PPIase, for example, from mammal which is constituted from a large number of cells endowed with highly differentiated functions, may differ from that, for example, from yeast which is unicellular organism and Escherichia coli which is prokaryote, is believed to be a logical conclusion in view of the different functions to be fulfilled by the different cells mentioned above. The hypothesis that the action of CsA is modified through the inhibition of the PPIase activity has been proposed on the basis of the inhibitory effect of CsA on the porcine PPIase. To justify the hypotecis, it is important to clarify the question whether or not the activities of PPIases from many organisms of widely diverging phylogenic origins, for example, Escherichia coli and yeast, are inhibited by CsA. Moreover, it is not correlated yet between the distribution of the CsA-binding activity in lower organism and the inhibitory effect of CsA on their PPIases. The presence of multiple forms of PPIases in various cells suggest that each PPIase acts on its specific substrates in each cell. This point, coupled with the finding that a cell specific form PPIase may be present in the photoreceptor cells of Drosophila evidently acquires a profound significance in the use of a recombinant DNA for the production of a specific protein with a specific cell as a host. It is desirable that PPIases which affect the folding of the aimed protein or the process of the protein synthesis thereof, coexist with the aimed protein produced in the host cell. Generally, it is considered that the PPIase of the host cell effectively acts on the protein inherent in the host cell. Therefore, the PPIases and their genes of the Escherichia coli and yeast, which are frequently used as hosts for the production of useful substances by the recombinant DNA technology, may be useful for the purpose mentioned above. Various organisms are being studied for the presence of cyclophyllin, using as an index the protein's ability to bond cyclosporin A, and the cyclosporin A bonding activity has been detected in arthropoda (cockroaches), trematoda, mollusks, molds, porifera, yeasts, and plants (pumpkins) as well as in mammals [Koletsky, A.J., Harding, M.W. & Handschumacher, R.E. J. Biol. Chem. 137, 1054-1059, 1986]. Nevertheless, no correspondence has been established between these activities and the PPIase activity. Though it has been demonstrated that the porcine PPIase accelerates the folding of protein, the question of whether or not the PPIase is actually present and exhibits the activity in the species such as insert, yeast and microbacteria etc., and the question of the extent to which it participates in the folding of protein, have not been definitely answered. As one of the means for solving the numerous problems mentioned above, it is believed important to isolate PPIases from Escherichia coli and yeast, study the nature of the PPIases as protein, and obtain the genes thereof. As described above, examples have been found in which PPIases are associated with the folding of proteins. In these cases, however, only one species of PPIase has been identified for one species of organism, and thus the utility of the PPIase for the folding of protein has been limited. This invention, therefore, is directed to providing a species of enzyme homogeneously isolated from Escherichia coli and is further directed to providing genes coding for the enzyme and a method of reproducing the enzyme in a high yield by using the gene. The present invention provides an E . coli PPIase-β characterized by possessing the following properties: (1) acting on and isomerizing the peptide bond X aa -Pro (wherein X aa stands for any amino acid and Pro for L-proline), (2) exhibiting a single molecular weight of about 20,000 daltons in the sodium dodecyl sulfatepolyacrylamide gradient gel electrophoresis, (3) exhibiting a single isoelectric point of about 5.0 in the isoelectric focusing, and (4) not being inhibited by CsA; a method of producing the enzyme characterized by recovering from E . coli cells; a gene coding for the enzyme; and a method of producing the enzyme by using the gene. In the drawings: Fig. 1 shows an elution profile in separation of E. coli extract, using a DEAE-Sepharose CL-6B column; Fig. 2 shows an elution profile in purification by gel filtration of the main fraction (β-fraction) shown in Fig. 1 with a Sephadex-G75 column, wherein _ _ • _ indicates the elution of protein detected by absorbance at 280 nm and _ ο _ indicates the elution o the PPIase activity. Fig. 3 shows result of determination of molecular weights of purified PPIase-α and PPIase-β; Fig. 4 shows result of determination of isoelectric points of purified PPIase-α and PPIase-β; Fig. 5 is a diagram showing that the purified PPIase-β gives a single peak in analysis with the reverse-phase Aquapore RP-300 column; Fig. 6 shows result of separation of peptide fragments obtained from the trypsin digestion of PPIase-β using a reverse-phase Spher 56P-18 column; Fig. 7 shows result of separation of peptide fragments obtained from the cyanogen bromide cleavage of PPIase-β using a reverse-phase Aquapore RP-300 column; Fig. 8 is a graph showing an effect of CsA on the activities of yeast and E. coli PPIase of the present invention, compared with porcine PPIase activity, in term of the relationship between CsA concentration and residual activity of PPIase; Fig. 9-1 and Fig. 9-2 respectively (SEQ ID NO 19), show a nucleotide sequence of a gene derived from E. coli and coding for the PPIase-β of the present invention, and a deduced amino acid sequence therefrom; Fig. 10 shows a process for construction of a plasmid pBLΔEPPIb; Fig. 11-1 (A) shows a process for construction of an expressing plasmid pATtrp EPPIb, and Fig. 11-2 (B) (SEQ ID NO 20) shows a nucleotide sequence of a linking region between trpE promoter and PPIase gene in plasmid pATtrp EPPIb; Fig. 12 shows an expression profile of the PPIase-β produced by transformed E. coli cells; Fig. 13 shows an elution profile in separation of PPIase-β produced by the transformed E. coli cells using a DEAE-Sepharose CL-6B column; Fig. 14 shows an elution profile in purification by gel filtration of a fraction obtained in Fig. 13 on a Sephadex-675 column; Fig. 15 shows the comparison between a natural PPIase-β and recombinant PPIase (β) in SDS-electrophoresis; Fig. 16 shows the comparison between a natural PPIase-β and recombinant PPIase (β) of their isoelectric point by the isoelectric focusing; Fig. 17 shows the comparison between a natural PPIase-β and recombinant type PPIase (β) on a reverse-phase Aquapore RP-300 column; Fig. 18 shows the comparison among natural PPIase-α, natural PPIase-(β), and the recombinant PPIase-β for their activities; Fig. 19A and 19B (SEQ ID NOS 21-33) shows the comparison of amino acid sequences of PPIases or cyclophyllin from various origins; and, Fig. 20 shows hydropathy patterns of amino acid sequences of PPIases of various origins. The recovery of the PPIase from E. coli can be accomplished by any of methods conventionally used for obtaining an enzyme from microorganisms. Any of the known strains, e.g., E . coli ST 249 strain [Kajie, S., Miki, K., Lin, E.C.C. & Anraku, Y. FEMS Microbiol. Lett., 24 , 25-29 (1984)] may be used as the starting material. Two PPIase components, i.e., PPIase-β as a main component and PPIase-α as a minor component, are obtained. These PPIases are treated with trypsin and cyanogen bromide, the amino acid sequences of the fragments determined, and the N-terminal amino acid sequences determined by a conventional method, to characterize PPIases in amino acid sequences. An oligonucleotide probe is designed on the basis of the information from the amino acid sequences of the PPIase-β protein. Then, a gene coding for PPIase of the present invention is obtained by screening a DNA library from E . coli with the said oligonucleotide probe. A nucleotide sequence of the cloned DNA is determined. The agreement between the corresponding part of the amino acid sequence estimated from the above nucleotide sequence and the amino acid sequence of the tryptic fragment of the protein (β) isolated from E . coli demonstrates that the DNA derived from E . coli encode the E . coli PPIase-β. Further, the comparison of the determined amino acid sequence with the amino acid sequences of the known PPlases demonstrates that the PPIase-β is a novel enzyme different from the known PPIases. The PPIase-β of the present invention possesses the amino acid sequence shown in Fig.9 SEQ ID NO 19). The present invention, however, is not limited thereto, but involves enzymes wherein one or more amino acids in the amino sequence shown in Fig. 9 (SEQ ID NO 19) have been substituted with other amino acids, and/or one or more amino acids have been added to said amino acid sequence, and/or one or more amino acids have been removed from said amino acid sequence, and the same characteristics as E . coli PPIase-β are still retrained. The characteristics of the enzyme of the present invention will be described in detail in Example 2. The PPIase of the present invention catalyzes the isomerization of prolyl peptide bond in protein and accelerate the folding of the protein, and therefore, can be used for converting a physiologically active polypeptide in inactive form produced by genetic manipulation into a correct conformation of active form. Further, the physiologically active polypeptide of interest can be produced directly in the active form by inserting a gene coding for the PPIase of this invention in an expressible form to a host to which had been inserted a gene coding for a physiologically active polypeptide in an expressible form, and expressing the two genes in the same host. Wherein two different kinds of enzyme are provided within one species, the different enzymes suitable for the activation by accelerating the folding of different proteins within the same species, can be made available independently. Since the PPIase of the present invention does not interact with CsA, it is useful as a reagent for studying the mechanism of action of CsA in comparison with PPIase from mammal or yeast on inhibition by CsA, and as other research reagents. The gene coding for PPIase of the present invention, is useful for the production of the PPIase of the present invention by the genetic manipulation, as well as in a modified form, useful as a gene material for the production of PPIase derivatives in which the enzymatic activity, stability, intracellular localizability, and substrate specificity thereof are altered. Moreover, the gene of the present invention is useful as a probe for screening a gene coding for other PPIase enzyme. When the aforementioned gene is used to produce a PPIase in microorganism host, such as recombinant Escherichia coli, as described in Examples 4 to 6, the PPIase can be produced much more efficiently than that extracted from non-recombinant E. coli. The PPIase thus produced using the recombinant possesses the same characteristics as the PPIase extracted from non-recombinant E.coli . as described in Example 8. Examples For a more specific illustration of the present invention, the following examples are presented. These are intended to be merely illustrative of and do not in any sense limit the invention. Example 1 Purification of E.coli PPIase In 500 ml of a 0.1M Tris-HCl buffer (pH 7.8) containing 5 mM 2-mercaptoethanol, 200 g of an Escherichia coli (ST 249 strain) cultured under anaerobic conditions was suspended, repeated two cycles of freezing and thawing treatments, and then stirred with Polytron. The suspension was treated with 2 g of lysozyme at room temperature for 30 minutes, stirred with Polytron, and centrifuged. To the supernatant was added ammonium sulfate to 80% saturation, with left standing at 4°C overnight. The resulting precipitate was collected by centrifugal separation, suspended in 10 mM Tris-HCl buffer (pH 7.8) containing 0.05% NaN 3 , dialyzed against the same buffer and then subjected to centrifugal separation. After removal of the precipitate in the dialysate, the supernatant was applied on an ion-exchange DEAE-Sepharose CL-6B column (2.5 cm in diameter x 40 cm). The peptidyl prolyl cis-trans isomerase (PPIase) activity was measured by the method described in Takahashi, N. et al., Nature, 337, 473-475 (1989). The elution of the sample from the column was effected by increasing sodium chloride concentrations by stepwise to 0.05 M, 0.1 M, 0.2 M, 0.3 M, and 0.5 M. The elution was carried out at a flow rate of 20 ml/hr. The elution profile obtained from the DEAE column chromatograph is shown in Fig.1 The E. coli PPIase activity was detected in the pass through fraction (salt concentration, 0 M) and the fraction eluted with a salt concentration of 0.1 M. Of the total amount of activity, about 10% was obtained in the pass through fraction and about 90% in the fraction eluted with a salt concentration of 0.1 M. The component in the pass through fraction is referred to as a minor component (α) and that in the fraction eluted with a salt concentration of 0.1 M as a main component (β). The two fractions were respectively purified by gel filtration on a Sephadex-G75 column (2.5 cm in diameter x 90 cm). The purification process of the main fraction is shown in Fig. 2. A 10 mM Tris-HCl buffer (pH 8.0) containing 0.15 M NaCl and 0.05% NaN 3 was used as a eluent, and a flow rate was 10 ml/hr. The main component of PPIase was purified by this purification step to a single component as determined by purity tests on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and reversed-phase high performance liquid chromatography. The main component is referred to as PPIase-β. The minor component is referred to as PPIase-α. Example 2. Characterization of Escherichia coli PPIase-β (1) Determination of molecular weight The molecular weight of the PPIase-β obtained in Example 1 was determined by the SDS-polyacrylamide concentration gradient gel (12% - 30% polyacrylamide gel) electrophoresis. As molecular weight standards, phosphorylase (molecular weight: 94,0000, bovine serum albumin (67,0000, ovalbumin (43,000), carbonic anhydrase (30,000), soybean trypsin inhibitor (20,100), and α-lactalbumin (14,000) were used. The PPIase-β was found to possess a single molecular weight of about 20,000 daltons as shown in Fig. 3. (2) Determination of isoelectric point The isoelectric point of the PPIase-β obtained in Example 1 was determined by the isoelectric focusing according to Amphline Isoelectric Focusing Manual (LKB Corp), using cytochrome C (pI 10.6), whale myoglobin (8.30), equine myoglobin (7.3), porcine myoglobin (6.45), porcine trifluoroacetyl myoglobin (5.92), azurin (5.65), C-phycocyanine (4.85), and C-phycocyanine (4.65) as standards. The PPIase-β of this invention was found to have a single isoelectric point of about 5.0, as shown in Fig. 12. (3) Homogeneity in reverse-phase column The PPIase-β of the present invention was applied on a reverse-phase Aquapore RP-300 column (2.1 mm in diameter x 3 cm; produced by Applied biosystems Corp), and eluted with 0.1% trifluoroacetic acid containing acetonitrile in a linear gradient concentrations of 0% to 100% over a period of 45 minutes at a flow rate of 200 µl/min. The PPIase gave a single peak, as shown in Fig. 5. . (4) Determination of partial amino acid sequence Amino acid sequences of tryptic fragments A solution of 200 µg of E. coli PPIase-β dissolved in 50 µl of 0.1 M of NH 4 HCO 3 ,was digested with 4 µg of TPCK-treated trypsin at 37°C for 8 hours. The digest was separated and purified on a reversed-phase Spher 5RP-18 column (2.1 mm in diameter x 3 cm) and Aquapore RP-300 column (2.1 mm in diameter x 3 cm) (both from Applied Biosystems). In this case elution was performed with a linear gradient from 0% to 100% of acetonitrile concentration in 0.1% trifluoroacetic acid or 0.1% heptafluorobutylic acid over a period of 45 minutes at a flow rate of 200 µl/min, to obtain 18 peptide peaks. The elution profile is shown in Fig. 6. Seven of the peptide fragments were analyzed to determine the amino acid sequence with an automatic sequencer model 477A (Applied Biosystems), and as a result, the following amino acid sequences were obtained: Amino acid sequences of fragments resulting from c yanogen bromide cleavage In a 70% formic acid, 200 µg of the PPIase was hydrolyzed by treatment with 500 µg of cyanogen bromide overnight. The resultant fragments were separated and purified on reverse-phase Spheri 5RP-18 (2.1 mm in diameter x 3 cm) and Aquapore RP-300 (2.1 mm in diameter x 3 cm) columns (both from Applied Biosystems). In this case, Elution was carried out by a linear gradient of 0% to 100% acetonitrile concentration in 0.1% trifluoroacetic acid or 0.1% heptafluorobutylic acid over a period of 45 minutes at a flow rate of 200 µl/minute to obtain 15 peptide peaks. The elution profile is shown in Fig. 7. Six of these peptide fragments were analyzed for amino acid sequence with an automatic sequencer, model 477A (Applied Biosystems). As the result, the following amino acid sequences were obtained: (5) N-terminal amino acid sequence The PPIase-β was analyzed for amino terminal sequence using an amino acid sequencer, model 477A (from Applied Biosystems). As the result, the following sequences were obtained: (6) Amino acid composition To 5 µg of the purified PPIase-β in a test tube was added 0.5 ml of 6 N HCl, and the mixture was degassed, sealed in tube, and hydrolyzed at 110°C for 24 hours. The resultant hydrolyzate was evaporated under a vacuum, and analyzed for amino acid composition using an amino acid analyzer, JLC-300 (Japan Electron Optics Laboratory Co., Ltd.). The results are shown in Table 1. Amino acid PPIase-β (Molar ratio) PPIase-α (Molar ratio) Recombinant PPIase-β (Molar ratio) Asp 22.23 (23) 26.2 22.6 Thr 11.36 (12) 10.45 11.2 Ser 5.41 (6) 7.91 5.8 Glu 18.50 (17) 17.91 16.5 Gly 16.00 (16) 15.80 14.2 Ala 10.00 (10) 14.43 10.0 Cys - (2) - - Val 11.64 (15) (2) 14.84 13.0 Met 5.35 (6) 3.94 5.3 Ile 8.75 (10) 8.38 8.3 Leu 5.23 (5) 8.87 5.6 Tyr 3.04 (3) 4.33 2.7 Phe 11.94 (12) 11.00 11.6 His 4.98 (5) 4.59 5.0 Lys 11.33 (11) 13.85 10.7 Arg 5.00 (5) 5.52 4.9 Pro 4.04 (5) 12.35 2.6 Trp - (1) - - (7) Sensitivity of Escherichia coli PPIase-β to cyclosporin A (CsA) The inhibition effect of CsA on the activity of purified E. coli PPIase-β was determined by the method described in N. Takahashi et al, Nature , 377 , 473-475 (1989). The results are shown in Fig.8. The figure shows the comparison of the degree of inhibition by cyclosporin A on Escherichia coli PPIase and the porcine PPIase. The cyclosporin A in a concentration enough to inhibit completely the porcine PPIase and yeast PPIase, brings virtually no inhibition of the Escherichia coli PPIase-β. The binding of CsA inhibits porcine PPIase and yeast PPIase activities, therefore, a positive interrelation appears to exist between the binding activity of CsA to the PPIase and the inhibition effect of CsA on the PPIase activity. It is assumed that the effects of CsA on various organisms, such as antifungal effect, antischistosome effect, antimalarial effect and the like, are manifested through the binding activity of CsA to PPIase, namely the inhibition effect of CsA on PPIase activity. Note, the fact that the inhibition effect of CsA is not observed on E . coli PPIase agrees with the fact that the binding activity of CsA present in all of tested eukaryotes was not detected in E. coli. Example 3 Cloning of Escherichia coli PPIase-β gene High-molecular-weight DNA was prepared from E . coli HB101 strain, according to the method described by Thomas et al. [ J. Mol. Biol, 11 , 476 (1965)]. About 2 µg of the high molecular DNA was digested in 20 µl of a solution [10 mM Tris-HCl (pH 7.5), 100 mM NaCl, 6 mM MgCl 2 , 6 mM mercaptoethanol, 0.1% gelatin, 10 units of Bgl II (Nippon Gene K.K.), and 10 units of Hind III (Nippon Gene K.K.)] at 37°C for 3 hours, subjected to 0.8% agarose gel electrophoresis, and transferred onto a membrane filter (Amersham Corp., "Hybond-N") according to the Southern method [Southern, E. M., J. Mol. Biol. , 98 , 503 (1975)]. The DNA was fixed on the filter by exposure to UV light for 2 minutes and then hybridized with a probe as follows. As the probe, the DNA chain: (SEQ ID. No. 15) synthesized by an automatic DNA synthesizer (Applied Biosystems Corp., "Motel 380") on the basis of an amino acid sequence from methionine at 59 position to proline at 67 place position in E . coli PPIase-β. The synthetic DNA (20 p moles) was reacted in 50 µl of a solution containing 50 mM Tris-HCl (pH 7.5), 10 mM MgCl 2 , 5 mM dithiothreitol, 100 µ Ci [γ- 32 P] ATP (3000 Ci/mmol, from Amersham Corp.), and 12 units of T4 polynucleotide kinase (produced by Takara Shuzo Co., Ltd.) at 37°C for 60 minutes, to label 5'-end with 32 P. The filter was immersed in a prehybridization solution [5 x Denhardt solution (100 x Denhardt solution = 2% bovine serum albumin, 2% Ficoll, and 2% polyvinylpyrrolidone), 1 M NaCl, 50 mM Tris-HCl (pH 7.5), 10 mM EDTA (pH 8.0), 0.1% sodium dodecyl sarcosinate, and 20 µg/ml of sonicated salmon sperm DNA] at 37°C for 1 hour, and then in a hybridization solution [a solution obtained by incorporating in the prehybridization solution about 10 6 cpm/ml of the aforementioned labeled DNA] at 37°C for 15 hours. The filter was washed with a 6 x SSC (20 x SSC = 3 M NaCl, 0.3 M trisodium citrate) solution at room temperature, further washed with a solution of 3 x SSC and 0.1% sodium dodecyl sarcosinate at 37°C for 30 minutes, and exposed to X-ray film (Kodak, "XAR-5") at -80°C for 4 days. The developed film showed that about 1 kb of Bgl II/Hind III DNA fragment was hybridized with the probe. Thus, an E . coli gene library containing about 1 kb DNA fragments was prepared as follows. About 50 µg of the aforementioned E . coli high-molecular-weight DNA was digested in a solution [10 mM Tris-HCl (pH 7.5), 100 mM NaCl, 6 mM MgCl 2 , 6 mM mercapto ethanol, 0.1% gelatin, 200 units of a Bgl II (Nippon Gene K.K.), and 200 units of a Hind III (Nippon Gene K.K.)] at 37°C for 3 hours, and subjected to 0.8% agarose gel electrophoresis. An about 1 kb DNA fragment was separated and purified by the glass powder method (Bio-101 Corp., "Gene Clean™"). About 50 ng of the recovered BgI II/Hind III DNA fragment and about 100 ng of pUC119 vector digested with BamHI and Hind III were ligated in 40 µl of a DNA ligation kit solution A and 8 µl of a solution B (Takara Shuzo Co., Ltd.) at 16°C for 15 hours, to obtain a recombinant plasmid 20 µl of the resultant reaction mixture was used to transform 400 µl of competent JM 109 strain cells (Epicurian coli™, STRATAGENE) in accordance with the manufacture's specification. The transformants were spread on ten X-Gal plates with a diameter of 90 mm [50 µg/ml of 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside, 80 µg/ml of isopropyl-β-D-thiogalactopyranoside, 25 µg/ml of ampicillin, an LB culture medium (1% bactotrypton, 1% NaCl, 0.5% yeast extract), and 1.5% agar]. These plates were incubated at 37°C overnight, to obtain about 1,500 transformants as white colonies. They were used as a library for the screening of E . coli PPIase genes. The recombinant colonies were transferred onto a filter (Amersham Corp., "Hybond-N"). The filter was set for 5 minutes on a 3MM filter paper (Whatman Corp.) impregnated with 0.5N NaOH and 1.5 M NaCl, with a colony-deposited surface of the filter facing upper side, and then for 5 minutes on the same filter paper impregnated with 1 M Tris-HCl (pH 7.5) and 1.5 M NaCl. Then, the filter was washed with a 2 x SSC solution, air dried, and irradiated with UV light to fix E . coli DNA on the filter. The filter thus obtained was subjected to hybridization under the same conditions as described above using a synthetic DNA as a probe. Then, the filter was washed and exposed to X-ray film and developed. Nine positive clones obtained were used for subsequent analysis. Specifically, each of the colonies of interest on the plates was inoculated to an LB culture medium containing 25 µg/ml of ampicillin, and shaken cultured at 37°C overnight. 1.5 ml of the resultant culture was used to prepare a recombinant DNA by the alkali bacteriolytic method [Ish-Horowicz, D. & J.F. Burke, Nucleic Acids Res. , 9 , 2989 (1981)]. The 9 clone DNAs thus obtained were subjected to double digestion with EcoRI and Hind III, then to 1% agarose electrophoresis, and further to Southern analysis as described above. As a result, only one clone containing 950 bp of E. coli DNA fragment was found to be a positive clone. This plasmid DNA was designated as pEPPIb. The insert of pEPPIb was subcloned to a M13mp type phage DNA and analyzed by the dideoxy method to determine its nucleotide sequence (Fig. 9 (SEQ ID NO 19)). An amino acid sequence estimated from the nucleotide sequence was completely identical with the amino acid sequence of peptide obtained from the E. coli main component (PPIase-β), therefore, the isolated gene was established as a gene coding for E. coli PPIase-β. This gene coded for a protein consisted of 164 amino acid residues and possessing a molecular weight of 18,184 daltons as calculated from the amino acid sequence. This molecular weight agrees very closely with that estimated from the data of SDS electrophoresis. Moreover, the amino acid composition agrees very closely with the found value (Table 1). Fig. 19 (SEQ ID NOS 21-33) shows the total amino acid sequence of E . coli PPIase-β and partial amino acid sequence of PPIase-α as compared with the sequences of the other cyclophilins. In the figure, amino acids conserved among all species, including E . coli , in homologous regions are within boxes. The amino acid sequence of E . coli PPIase-β shows about 25% of homology to those of cyclophilin/PPIases from the other species, and especially high homology in regions from the 30th to 70th residues and from the 100th to 120th residues as counted from the amino terminal side was observed. Also E . coli PPIase-α shows a partially high homology to PPIase-β and conserved amino acid residues in highly homologous regions among the sequences of the other species. Therefore, both of PPIase-β and -α from E . coli appears to be cyclophilin-like PPIases. Unlike the cyclophilin/PPIases from the other species, specifically eukaryotes the PPIases from E . coli possess no sensitivity to CsA. The comparison of their structures is believed to offer the basis for the elucidation of the CsA binding pattern of other species, and of the mechanism of action of PPIases as enzyme, and the like. In the case of porcine PPIase, an SH modifying reagent induces modification of four cysteine residues and deprives its enzymatic activity. However, the modification of one of cysteine is inhibited, when CsA binds to the enzyme. After this modification, the enzymatic activity is recovered by releasing CsA from the enzyme by dilution. It has been considered, therefore, that CsA protects the cysteine residue from the modification with the SH reagent by directly interacting with the cysteine residue as active group [Fisher, G., Wittmann-Liebold, B., Lang., K., Kekfhaber, T. & Schmid, F.X. Nature 337 , 476-478 (1989)]. On the other hand, the PPIases derived from E. coli have affected on their enzymatic activity neither by the CsA nor by the SH modifying reagent. Looking at the amino acid sequence in Fig. 19 (SEQ ID NOS 21-33 from this point of view, no cysteine residue is found to be perfectly conserved among all of PPIases from the given organisms. In the comparison of hydropathy patterns [Kyte, J.M. & Doolittle, R.F., J. Mol. Biol. 157 , 105-132 (1982)] of porcine and yeast PPIases demonstrated to bind CsA and to be inhibited by CsA on the enzymatic activities (see our previous patent application related to yeast PPIase) with E . coli PPIase-β demonstrated to be not inhibited by CsA, Cys-115 in porcine and Cys-117 in yeast which are common to both porcine and yeast PPIases sensitive to CsA and absent from E . coli , are located at similar positions on hydropathy patterns (Fig. 20). No cysteine residues are present at such positions in E . coli PPIase-β, and therefore, Cys-115 in porcine and Cys-117 in yeast are predicted to interact directly with CsA. In the light of the data for E . coli , the theory that the above cysteine residues are active groups directly associated with the expression of enzymatic activity leaves room for doubt. At least in the case of E . coli, no proof has been offered that the cysteine residues are active groups. Rather, the two high homology regions found among the amino acid sequences from all species, are believed to be important for the expression of the activities. Note, Escherichia coli HB101/pEPPIb containing plasmid pEPPIb has been deposited at the Fermentation Research Institute, Agency of Industrial Science and Technology under FERM P-11042. Example 4 Construction of plasmid pBLEPPIb ( Fig. 10). In 100 µl of a solution [50 mM Tris-HCl (pH 7.6), 7 mM MgCl 2 , 150 mM KCl, and 20 units of PvuI (produced by Nippon Gene K.K.)], 6 µg of a plasmid (pEPPIb) containing E . coli PPIase-β gene was digested at 37°C for 180 minutes. The digest was extracted twice with 50 µl of a phenol/chloroform (1 : 1) mixture to remove proteins, and precipitated with ethanol. The recovered DNA was dried with a centrifugal evaporator, dissolved in 100 µl of a Mung bean nuclease buffer (Takara Shuzo Co. Ltd., "DNA deletion kit"). After an addition of 2 µl of Mung bean nuclease (Takara Shuzo Co., Ltd., "DNA deletion kit"), the mixture was allowed to react at 37°C for 60 minutes, extracted twice with 50 µl of a phenol/chloroform (1 : 1) mixture to remove proteins, and precipitated with ethanol. The recovered DNA was dried with a centrifugal evaporator, reacted with 30 µl of a solution [100 mM Tris-HCl (pH 7.6), 7 mM MgCl 2 50 mM NaCl, and 20 units of EcoRI (Nippon Gene K.K.)] at 37°C for 60 minutes, and subjected to 1.2% agarose gel electrophoresis. The inserted DNA was recovered by the glass powder method (Bio-101 Corp., "Gene Clean™"), and extracted with 10 µl of a TE solution [10 mM Tris-HCl (pH 7.5) and 1 mM EDTA]. 4 µl of the inserted DNA was ligated with about 15 ng of a vector DNA (Bluescript II SK+) previously digested with the restriction enzymes, EcoRI (produced by Nippon Gene K.K.) and EcoRV (produced by Nippon Gene K.K.), in 30 µl of DNA Ligation kit solution A (Takara Shuzo Co., Ltd.) and 5.5 µl of solution B at 16°C for 16 hours, to obtain a recombinant plasmid. Escherichia coli XL-1 Blue was transformed with 20 µl of the ligation mixture by the calcium chloride method [Mandel, M & Higa, A. (1970) J. Mol. Biol. 53, 149]. The transformant was selected as white colonies obtained by culturing the cells on an X-Gal plate (155 µg/ml 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside, 80 µg/ml isopropyl-β-D-thiogalactopyranoside, 50 µg/ml ampicillin, 1% NaCl, 0.5% yeast extract, 1% trypton, and 1.5% agar) at 37°C for 16 hours. The transformant was inoculated to an LB culture medium (1% NaCl, 0.5% yeast extract, and 1% trypton) containing 50 µg/ml of ampicillin, and cultured therein at 37°C for 6 hours. From the culture, a plasmid DNA was prepared by the alkali bacteriolytic method [Birnboim, H. C. & Doly. J. (1979) Nucleic Acids Res. 7 , 1513], dissolved in 30 µl of a TE solution. One third of DNA thus obtained was treated with a solution [50 mM Tris-HCl (pH 7.6), 7 mM MgCl 2 100 mM NaCl, 25 ng/µl ribonuclease A, 5 units of Xho I (Takara Shuzo Co., Ltd.), and 5 units of Bam HI (Nippon Gene K.K.)] at 37°C for 60 minutes, and then subjecting to 0.8% agarose gel electrophoresis, to confirm a plasmid containing the desired insert. The transformant was cultured in 200 ml of an LB medium containing 50 µg/ml of ampicillin and subjected to the alkali bacteriolytic method, to prepare a plasmid DNA. The plasmid DNA was further purified by the cesium chloride density gradient centrifugation [Maniatis, T. et al. (1982) Molecular Cloning: A Laboratory Manual, Cold Spring Habor Laboratory]. This plasmid was designated as pBLEPPIb. Example 5 Construction of plasmid pBLΔEPPIb (Fig. 11) . 29 µg of PBLEPPIb was digested in 1 ml of a solution [50 mM Tris-HCl (pH 7.6), 7 mM MgCl 2 , 150 mM NaCl, and 200 units of Sal I] at 37°C for 12 hours, and then left to stand at 5°C for 45 minutes to inactivate the restriction enzymes. To 500 µl of the resultant solution were added 10 µl of 1 mM Thio-dNTP (Stratogene Corp.; EXO/MUNG deletion kit) and 10 µl of Klenow fragment (Takara Shuzo Co., Ltd.), the mixture was allowed to react at 37°C for 45 minutes, to blunt the cleavage sites. The DNA solution was extracted with a phenol/chloroform (1 : 1) mixture to remove proteins, and precipitated with ethanol to recover the DNA. Next, this DNA was digested in 25 µl of a solution [50 mM Tris-HCl (pH 7.6), 7 mM MgCl 2 60 mM NaCl, and 40 units of Hind III]. phenol extracted, ethanol precipitated, and dried by centrifugal evaporator. The DNA thus obtained was dissolved in 100 µl of an Exo III buffer (Takara Shuzo Co., Ltd.; DNA deletion kit), reacted with 1 µl of exonuclease III (the same kit) at 23°C. In the course of this reaction, 10 µl of sample were taken up at intervals of 20 seconds, mixed with 10 µl of Mung mean nuclease buffer, and placed on ice to stop the reaction. A total ten samples thus obtained were combined, heated at 65°C for 5 minutes to inactivate exonuclease III, and then treated with 2 µl of Mung bean nuclease (produced by the same manufacturer) at 37°C for 60 minutes to create a DNA deletion. The mixture was phenol extracted and ethanol precipitated to recover the DNA. One quarter of the DNA thus recovered was treated with 100 µl of DNA Ligathion kit solution A and 12 µl of solution B (both from Takara Shuzo Co., Ltd.) at 16°C for 120 minutes, to cyclize the DNA. The cyclized DNA was digested with Sal I to remove unreacted plasmid DNA. The obtained plasmid DNA was used to transform the E . coli XL-1 Blue strain by the CaCl 2 method. A plasmid DNA was purified in the same manner as described above from the transformant, double digested with Xho I (Takara Shuzo Co., Ltd.) and EcoRI Nippon Gene K.K.), and then subjected to 1.2% agarose gel electrophoresis, to select a transformant having an insert with a desired length. The 5'-terminal base sequence of the PPIase-β gene in plasmid DNA molecule from the transformant was determined by using a M13 Sequencing kit (Toyobo K.K.). One clone believed to be most suitable for the construction of the PPIase-β gene expressing plasmid, was selected. This plasmid was designated as pBLΔEPPIb. Example 6 Construction of expression plasmid pATtrp EPPIb Ten µg of the pBLΔEPPIb plasmid DNA obtained in Example 5 was treated with 50 µl of a solution [50 mM Tris-HCl (pH 7.6), 7 mM MgCl 2 100 mM NaCl, and 20 units of Xho I (Takara Shuzo Co., Ltd.)] at 37°C for 120 minutes, phenol extracted, and ethanol precipitated to recover the DNA. This DNA was treated with 50 µl of Klenow buffer [7 mM Tris-HCl (pH 7.5), 7 mM MgCl 2 , 100 mM NaCl, 0.1 mM EDTA, and 5 units of Klenow fragment (Takara Shuzo Co., Ltd.)] at 37°C for 50 minutes, to blunt the Xho I cleavage sites, phenol extracted, and then ethanol precipitated to recover the DNA. The DNA thus obtained was further treated with 30 µl of a solution [50 mM Tris-HCl (pH 7.6), 7 mM MgCl 2 , 100 mM NaCl, and 20 units of BamH I (Nippon Gene K.K.)] at 37°C for 60 minutes, subjected to 1.2% agarose gel electrophoresis, and recovered the inserted DNA by the glass powder method. The inserted DNA was extracted from 10 µl of a TE solution. On the other hand, Pst I/Cla I double-digested pP L -TNF (Ikenaka et al., Chem. Pharm. Bulletin , in press) comprising a trp promotor trp LSD sequence, was ligated with a Pst I/Cla I large fragment from pAT153, to obtain pATtrp vector (disclosed in Japanese Patent Application No. 88-37,452) and 10 µg of the vector DNA was digested with Cla I (BioRad Co.), blunted the cleavage site with a Klenow fragment digested with a BamH I, and then subjected to 1.2% agarose gel electrophoresis to separate the inserted DNA. The inserted DNA was recovered by the glass powder method, and extracted from 10 µl of TE solution. Then, 2 µl of the DNA fragment containing E. coli PPIase-β gene obtained was ligated with 30 µl of a linearized pATtrp in 30 µl of a DNA Ligation kit solution A and 6 µl of solution B (both from Takara Shuzo Co., Ltd.) at 16°C for 16 hours, a cyclic recombinant plasmid was obtained. The expression plasmid was designated as pATtrp EPPIb. Escherichia coli HB 101 strain was transformed with using the expression plasmid pATrp EPPIb by the calcium chloride method. The obtained transformant was used for an experimental expression of E . coli PPIase-β in E . coli cells. In the following experiment, the transformant containing pATtrp plasmid was used as a control. Fifty µl of a glycerol stock of the transformant was inoculated to 5 ml of an LB culture medium containing 50 µg/ml of ampicillin, and cultured at 37°C overnight. Fifty µl of the preculture was inoculated to 100 ml of a M9CA-amp culture medium (0.05% NaCl, 0.6% Na 2 HPO 4 , 0.3% KH 2 PO 4 , 0.1% NH 4 Cl, 0.2% casamino acid, 2 mM MgSO 4 , 0.2% glucose, 0.1 mM CaCl 2 , 50 µg/ml ampicillin, pH 7.4) and shaken cultured at 37°C. When the turbidity (absorbance at 600 nm) of culture reached about 0.3, 3-β-indol-acrylic acid (IAA) was added to a final concentration of 50 µg/ml to induce expression, and the shaken culture was further continued at 37°C for about 20 hours. It was confirmed that the production of PPIase-β by the transformant was above 50 times more than that of the control, on quantitative determination by densitometry following electrophoresis (Fig.12). It is clear that the PPIase-β is expressed in a large amount in the soluble fraction of the transformant. Example 7 Production of PPIase-β To 5 ml of an LB-Am culture medium (10 g/ℓ bactotrypton, 5 g/ℓ yeast extract, 5 g/ℓ NaCl, and 50 µg/ml ampicillin) was inoculated 50 µl of the aforementioned transformant glycerol stock, and cultured at 37°C overnight. To 50 ml of an LB-Amp culture medium was inoculated 0.5 ml of the resultant culture and cultured again overnight. Then, 4 ml of the culture was inoculated to 400 ml of M9CA-Amp culture medium, cultured at 37°C for 2 hours, and after adding 0.8 ml of 20 mg/ml of 3-β-indolacrylic acid (IAA) solution, cultured further for 24 hours. The culture in 400 ml of M9CA-Amp medium was carried out in 10 replications. About 4 liters of combined culture was centrifuged at 6,000 rpm for 5 minutes at 4°C, to obtain 11.8 g of wet bacterial cells. 11.8 g of the bacterial cells were suspended in 50 ml of 0.1 M Tris-HCl (pH 7.5) solution containing 5 mM 2-mercaptoethanol, and treated with 11.8 mg of lysozyme at 30°C for 1 hour. The mixture was sonicated for 1 minute to homogenize cells, and centrifuged at 18,000 rpm for 30 minutes. This supernatant was dialyzed three times against 3 liters of 10 mM Tris-HCl solution (pH 8.0) containing 0.05% NaN 3 , and then applied on a DEAE-Sepharose CL-6B (2.5 cm in diameter x 40 cm) column previously equilibrated with the same buffer. The enzymatic activity of the peptidyl prolyl cis-trans isomerase was measured by the method described in Takahashi, N. et al, Nature , 337 , 473-475 (1989). The elution of the protein from the column was effected by stepwise elution of 0.05 M, 0.1 M, 0.2 M, and 0.3 M NaCl. The desired enzyme was detected in an eluate having a salt concentration of 0.1 M (Fig. 21). Fractions 176-196 having an enzymatic activity was concentrated with 80% saturated ammonium sulfate, and separated on a Sephadex-G75 column (2.5 cm in diameter x 90 cm) with 10 mM Tris-HCl (pH 8.02) containing 0.15 M NaCl and 0.05% NaN 3 (Fig.14), to obtain a purified sample. The fractions 64-78 shown in the figure were combined as a purified fraction, and characterized as described below. Example 8 Characteristic comparison of between recombinant Escherichia coli PPIase-β and natural countertype (1) Determination of molecular weight The molecular weights were determined by sodium dodecyl sulfate-polyacryl amide gradient gel (12% to 30% polyacrylamide gel) electrophoresis. As molecular weight standards, phosphorylase b (molecular weight: 94,000), bovine serum albumin (67,000), ovalbumin (43,000), carbonic anhydrase (30,000), soybean trypsin inhibitor (20,100), and α-lactalbumin (14,400) were used. The recombinant E. coli PPIase-P was found to possess a single molecular weight of 20,000 daltons as shown in Fig. 15, which could not be discriminated from that of natural countertype. (2) Determination of isoelectric point The isoelectric points were determined according to the Ampholline Isoelectric Focusing Manual published by LKB Corp. As standards, cytochrome C (pI 10.6), whale myoglobin (8.3), equine myoglobin (7.4), porcine myoglobin (6.45), porcine trifluoroacetyl myoglobin (5.92), azurin (5.65), and C-phycocyanine (4.85, 4.65) were used. The results are shown in Fig. 16. The recombinant E . coli PPIase-β was found to possess a single isoelectric point of about 6.2 as shown in the figure, which could not be discriminated from the natural countertype. (3) Comparison by reversed-phase column chromatography The sample was applied on a reversed-phase Aquapore RP-300 column (2.1 mm in diameter x 3 cm; Applied Biosystems Corp.). Elution was performed by a linear gradient of 0% to 100% acetonitrile concentration in 0.1% trifluoroacetic acid over a period of 45 minutes at a flow rate of 200 µl/minute. The purified enzyme gave a single peak. The retention time of the recombinant sample on the columns could not be discriminated from that of natural enzyme. The recombinant sample and the natural sample were mixed and subjected to separation. The mixture gave a single peak and failed to show any difference in behavior on the reversed-phase column (Fig. 17). (4) Amino terminal sequence When the standard PPIase obtained in Example 7 was analyzed with an automatic protein sequencer, Model 477A (produced by Applied Biosystems), the following sequence from the amino terminal methionine to the 20th residue was entirely identical with that of the natural enzyme: (5) Amino acid composition In a test tube, 20 µg of the purified PPIase was mixed with 0.5 ml of 6N HCl, degassed, sealed, and hydrolyzed at 110°C for 24 hours. The resultant hydrolyzate was dried under a vacuum, and then analyzed for amino acid composition with an amino acid analyzer, Model JLC-300 (NIPPON DENSI Co., Ltd.). The results are shown in Table 1. This table gives the composition values of recombinant and natural enzymes, indicating close agreement. (6) Determination of the specific activity of enzyme The specific activities of the recombinant and natural enzymes were calculated on the basis of the values obtained from the activity measurement according to the method described in N. Takahashi et al, Nature, 377, 473-475 (1989). As the result, the specific activity of the natural enzyme was 51,480 arbitrary units/A 200 1 unit, while that of the recombinant enzyme was 52,876 arbitrary units/A 280 1 unit. Thus, no distinction could be made on specific activity (Fig. 18). Deposition of microorganisms Escherichia coli JM107/pUC-y-PPI containing plasmid pUC-y-PPI has been deposited at the Fermentation Research Institute, Agency of Industrial Science and Technology (FRI), 1-3 Higashi 1-chome, Tsukuba-shi, Ibaraki, 305 Japan, as FERM P-10812 on July 4, 1989, and transferred to an international deposition under the Budapest treaty, as FERM BP-3002, on July 11, 1990. Escherichia coli HB101/pEPPIb containing plasmid pEPPIb has been deposited at the FRI as FERM P-11042 on October 3, 1989, and transferred to an international deposition under the Budapest treaty as FERM BP-3003 on July 11, 1990. SEQUENCE ID NO. 1 SEQUENCE TYPE Peptide SEQUENCE LENGTH 7 amino acids ORIGINAL SOURCE Escherichia coli (ST 249 strain) Enzyme PPIase-β FRAGMENT TYPE Tryptic Fragment IDENTIFIED E SEQUENCE ID NO. 2 SEQUENCE TYPE Peptide SEQUENCE LENGTH 11 amino acids ORIGINAL SOURCE Escherichia coli (ST 249 Strain) Enzyme PPIase-β FRAGMENT TYPE Tryptic Fragment IDENTIFIED E SEQUENCE ID NO. 3 SEQUENCE TYPE Peptide SEQUENCE LENGTH 17 amino acids ORIGINAL SOURCE Escherichia coli (ST 249 Strain) Enzyme PPIase-β FRAGMENT TYPE Tryptic Fragment IDENTIFIED E SEQUENCE ID NO. 4 SEQUENCE TYPE Peptide SEQUENCE LENGTH 12 amino acids ORIGINAL SOURCE Escherichia coli (ST 249 Strain) Enzyme PPIase-β (Beta) FRAGMENT TYPE Tryptic Fragment IDENTIFIED E SEQUENCE ID NO. 5 SEQUENCE TYPE Peptide SEQUENCE LENGTH 7 amino acids ORIGINAL SOURCE Escherichia coli (ST 249 Strain) Enzyme PPIase-β FRAGMENT TYPE Tryptic Fragment IDENTIFIED E SEQUENCE ID NO. 6 SEQUENCE TYPE Peptide SEQUENCE LENGTH 17 amino acids ORIGINAL SOURCE Escherichia coli (ST 249 Strain) Enzyme PPIase-β FRAGMENT TYPE Tryptic Fragment IDENTIFIED E SEQUENCE ID NO. 7 SEQUENCE TYPE Peptide SEQUENCE LENGTH 20 amino acids ORIGINAL SOURCE Escherichia coli (ST 249 Strain) Enzyme PPIase-β FRAGMENT TYPE Tryptic Fragment IDENTIFIED E SEQUENCE ID NO. 8 SEQUENCE TYPE Peptide SEQUENCE LENGTH 12 amino acids ORIGINAL SOURCE Escherichia coli (ST 249 Strain) FRAGMENT TYPE Enzyme PPIase-β Cyanogen bromide cleavage fragment IDENTIFIED E SEQUENCE ID NO. 9 SEQUENCE TYPE Peptide SEQUENCE LENGTH 9 amino acids ORIGINAL SOURCE Escherichia coli (ST 249 Strain) Enzyme PPIase-β FRAGMENT TYPE Cyanogen bromide cleavage fragment IDENTIFIED E SEQUENCE ID NO. 10 SEQUENCE TYPE Peptide SEQUENCE LENGTH 24 amino acids ORIGINAL SOURCE Escherichia coli (ST 249 Strain) Enzyme PPIase-β FRAGMENT TYPE Cyanogen bromide cleavage fragment IDENTIFIED E SEQUENCE ID NO. 11 SEQUENCE TYPE Peptide SEQUENCE LENGTH 27 amino acids ORIGINAL SOURCE Escherichia coli (ST 249 Strain) Enzyme PPIase-β FRAGMENT TYPE Cyanogen bromide cleavage fragment IDENTIFIED E SEQUENCE ID NO. 12 SEQUENCE TYPE Peptide SEQUENCE LENGTH 15 amino acids ORIGINAL SOURCE Escherichia coli (ST 249 Strain) Enzyme PPIase-β FRAGMENT TYPE Cyanogen bromide cleavage fragment IDENTIFIED E SEQUENCE ID NO. 13 SEQUENCE TYPE Peptide SEQUENCE LENGTH 11 amino acids ORIGINAL SOURCE Escherichia coli (ST 249 Strain) Enzyme PPIase-β FRAGMENT TYPE Cyanogen bromide cleavage fragment IDENTIFIED E SEQUENCE ID NO. 14 SEQUENCE TYPE Peptide SEQUENCE LENGTH 29 amino acids ORIGINAL SOURCE Escherichia coli (ST 249 Strain) FRAGMENT TYPE N-terminal IDENTIFIED E SEQUENCE ID NO. 15. SEQUENCE TYPE Nucleotide SEQUENCE LENGTH 26 bases SEQUENCE ID NO 16 SEQUENCE TYPE Peptide SEQUENCE LENGTH 20 amino acids ORIGINAL SOURCE Escherichia coli (ST 249 strain) Enzyme PPIase FRAGMENT TYPE N-terminal IDENTIFIED E SEQUENCE ID NO. 17 SEQUENCE TYPE Peptide SEQUENCE LENGTH 164 amino acids ORIGINAL SOURCE Escherichia coli peptidyl-prolyl cis-trans isomerase - β SEQUENCE ID NO. 18 SEQUENCE TYPE Nucleotide SEQUENCE LENGTH 492 bases ORIGINAL SOURCE Escherichia coli peptidyl-prolyl cis-trans isomerase - β SEQUENCE ID NO 19 SEQUENCE TYPE Nucleotide with corresponding protein SEQUENCE LENGTH 949 bases STRANDEDNESS Double ORIGINAL SOURCE E. coli MOLECULE TYPE Gene derived from E. coli and coding for the enzyme PPIase-β of the present invention IDENTIFIED Nucleotide sequence E; Amino acid sequence deduced from nucleotide sequence FEATURES Between bases 1 & 2 HindIII restriction site Between bases 47 & 48 EcoRV restriction site Between bases 348 & 349 PvuI restriction site Between bases 944 & 945 BglII restriction site Bases at 919 - 921 a stop codon SEQUENCE ID NO 20 SEQUENCE TYPE Nucleotide with corresponding amino acid sequence SEQUENCE LENGTH 21 bases STRANDEDNESS Double FEATURES PPIase-β gene initiation codon at base nos. 19, 20, 21 TrpE promoter down stream of base no. 4 SEQUENCE ID NO 21 SEQUENCE TYPE Protein SEQUENCE LENGTH 162 amino acids ORIGINAL SOURCE Porcine OTHER Amino acid sequence for comparison of PPIases or cyclophillin from various origins. SEQUENCE ID NO 22 SEQUENCE TYPE Protein SEQUENCE LENGTH 161 amino acids ORIGINAL SOURCE Bovine OTHER Amino acid sequence for comparison of PPIases or cyclophillin from various origins SEQUENCE ID NO. 23 SEQ TYPE Protein SEQ LENGTH 163 amino acids ORIGINAL SOURCE Human OTHER Amino acid sequence for comparison of PPIases or cyclophillin from various origins SEQUENCE ID NO. 24 SEQ TYPE Protein SEQ LENGTH 162 amino acids ORIGINAL SOURCE Rat OTHER Amino acid sequence for comparison of PPIases or cyclophillin from various origins SEQUENCE ID NO. 25 SEQ TYPE Protein SEQ LENGTH 170 amino acids ORIGINAL SOURCE Drosophila OTHER Amino acid sequence for comparison of PPIases or cyclophillin from various origins SEQUENCE ID NO. 26 SEQ TYPE Protein SEQ LENGTH 166 amino acids ORIGINAL SOURCE N. crassa OTHER Amino acid sequence for comparison of PPIases or cyclophillin from various origins SEQUENCE ID NO. 27 SEQ TYPE Protein SEQ LENGTH 160 amino acids IDENTIFIED E ORIGINAL SOURCE Yeast OTHER Amino acid sequence for comparison of PPIases or cyclophillin from various origins SEQUENCE ID NO. 28 SEQ TYPE Protein SEQ LENGTH 164 amino acids IDENTIFIED E ORIGINAL SOURCE E. coli PPIase-β OTHER Amino acid sequence for comparison of PPIases or cyclophillin from various origins SEQUENCE ID NO. 29 SEQ TYPE Peptide SEQ LENGTH 44 amino acids IDENTIFIED E ORIGINAL SOURCE E. coli PPIase-α FRAGMENT TYPE N-terminal OTHER Amino acid sequence for comparison of PPIases or cyclophillin from various origins SEQUENCE ID NO. 30 SEQ TYPE Peptide SEQ LENGTH 38 amino acids IDENTIFIED E ORIGINAL SOURCE E. coli PPIase-α FRAGMENT TYPE Internal OTHER Amino acid sequence for comparison from various origins SEQUENCE ID NO. 31 SEQ TYPE Peptide SEQ LENGTH 21 amino acids IDENTIFIED E ORIGINAL SOURCE E. coli PPIase-α FRAGMENT TYPE Internal OTHER Amino acid sequence for comparison of PPIases of cyclophillin from various origins SEQUENCE ID NO. 32 SEQ TYPE Peptide SEQ LENGTH 9 amino acids IDENTIFIED E ORIGINAL SOURCE E. coli PPIase-α FRAGMENT TYPE Internal OTHER Amino acid sequence for comparison of PPIases or cyclophillin from various origins SEQUENCE ID NO. 33 SEQ TYPE Peptide SEQ LENGTH 16 amino acids IDENTIFIED E ORIGINAL SOURCE E. coli PPIase-α FRAGMENT TYPE Internal OTHER Amino acid sequence for comparison of PPlases or cyclophillin from various origins

Claims: An Escherichia coli peptidyl prolyl cis.trans isomerase β possessing the characteristics: (1) acting on and isomerizing an X aa -Pro bond (wherein X aa stands for any amino acid and Pro for L-proline) in a peptide chain, (2) exhibiting a single molecular weight of about 20,000 daltons on sodium dodecyl sulfate-polyacrylamide concentration gradient gel electro-phoresis, (3) exhibiting a single isoelectric point of about 5.0 on isoelectric focusing, and (4) not being inhibited by CsA. The enzyme β according to claim 1, which possesses the following amino acid sequence: A method for producing an Escherichia coli peptidyl-prolyl cis-trans isomerase β, characterized by recovering the enzyme of claim 1 or claim 2 from cells of Escherichia coli. A gene coding for an Escherichia coli peptidyl-prolyl cis-trans isomerase β of claim 1 or claim 2. A gene according to claim 4, which possesses the following base sequence: An expression plasmid containing a gene of claim 4 or claim 5. A method of producing an Escherichia coli peptidyl-prolyl cis-trans isomerase β, characterized by culturing a host microorganism transformed with an expression plasmid of claim 6 and recovering an Escherichia coli peptidyl-prolyl cis-trans isomerase β from the resultant culture.

Inventors: HAYANO TOSHIYA, KATOU SETSUKO, MAKI NOBORU, SUZUKI MASANORI, TAKAHASHI NOBUHIRO, HAYANO, TOSHIYA, KATOU, SETSUKO, MAKI, NOBORU, SUZUKI, MASANORI, TAKAHASHI, NOBUHIRO, HAYANO, TOSHIYA, C/O TONEN CORPORATION, KATOU, SETSUKO, C/O TONEN CORPORATION, MAKI, NOBORU, C/O TONEN CORPORATION, SUZUKI, MASANORI, C/O TONEN CORPORATION, TAKAHASHI, NOBUHIRO, C/O TONEN CORPORATION
Applicants: TONEN CORP, TONEN CORPORATION
Application year: 1990
Main classification: C12N15
IPC codes: C12N9, C12N15