JPH03228675A - Escherichia coli bacterium peptidyl prolyl-cis-trans-isomerase - Google Patents

Abstract

PURPOSE:To make it possible to produce an uniformly isolated enzyme derived from Escherichia coli in large quantities by collecting Escherichia coli peptidyl prolyl-cis trans isomerase enzyme from a cultured material of a specific host microorganism. CONSTITUTION:The Escherichia coli peptidyl prolyl-cis-trans isomerase beta having the properties; (a) reacting with bond expressed by the formula in a peptide chain and isomerizing the peptide; (b) exhibiting single molecular weight of about 20000 dalton in dodecylsulfuric acid sodium salt polyacrylic amide concentration gradient gel electrophoresis; (c) exhibiting single isoelectric point of about 5.0 in isoelectric focusing method and (d) being not inhibited by cyclosporin A. A host microorganism transformed by manifestation plasmid containing a gene coding the above-mentioned enzyme beta is cultured and Escherichia coli peptidyl prolyl-cis trans isomerase beta is collected from the cultured mixture to provide the aimed isomerase.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention is directed to Escherichia colt.
The present invention relates to a peptidyl prolyl-cis-trans isomerase (PPIase) derived from this invention, a method for producing the same, and a gene encoding the enzyme.

PPIase has the function of promoting the formation of higher-order structure of proteins by catalyzing the isomerization of proline peptide bonds in proteins, and can be used as a means of activating inactive proteins produced by genetic engineering. It is useful as an analytical reagent, etc. In addition, the gene encoding the enzyme is useful for producing the enzyme through genetic engineering, and by modifying the gene through genetic engineering, the enzyme activity and intracellular localization can be improved. It is promising as a starting material for genes for producing enzyme derivatives with altered site, substrate specificity, stability, etc. Furthermore, the present invention provides a means for efficiently producing the useful protein having a desired higher-order structure by expressing the gene in the same host cell as the gene of the useful protein whose enzyme promotes higher-order structure formation.

[Conventional technology]

PPIase was found in pig kidney [Pzsher,
c, IBang, H, & Mech, C, Bi
omed, biochim, Acta 43
+1101-1111 (1984)), this enzyme isomerizes the Xaa-P, (Xaa represents any amino acid and P, represents L-proline) bond in oligopeptides. Bang+
H, & Mech, C, Biomed.

biochim, Acta 43.1101-111
1 (1984)], which is also known to promote the reconstitution of denatured immunoglobulin light chains and ribonuclease TI (Lang, K., Schmid,
F, & Fisher, G Nature, l1
9+268-270 (1987)). The amino acid sequence of this enzyme purified from pig kidney has been revealed.
It was found that this protein is the same as the protein (cyclophilin) that binds the immunosuppressant cyclosporin A (CsA), and that CsA inhibits PPIase activity. The effect of CsA is that the immunosuppressive effect is directly proportional to the ability of CsA to bind to cyclophilin, and that cyclophilin is the main component that binds CsA in immune cells.
It can be assumed that this is done through inhibition of the enzymatic activity of PPIase, which is the same as cyclophilin (
Takahashi, N. + Hayano, T.
&5uzuki, M., Nature 337.47
3-475 (1989)), but since CsA binding activity and PPIase activity have been found in almost all organisms and most organs of mammals, why is it that CsA used in organ transplantation does not bind immune cells, The mechanism of action, particularly whether it acts specifically on T cells, has not been explained.

Rhodopsin, a visual substance that exists in the retina of animals, is a protein in which opsin is bound to a chromophore, 11-C15-retinal.When exposed to the effects of light, the chromophore changes sequentially, and the maximum absorption wavelength changes. It becomes the starting material for the photoconversion process that changes accordingly. It is known that there is a mutant in Drosophila visual cells that inhibits the conversion of the precursor opsin to rhodopsin. Became. The results showed that the n1naA gene encodes a protein with amino acid sequence homology to cyclophilin. n1naA encodes a cyclophilin-like protein, and since cyclophilin is the same as PPIase, n1naA is a PPIase.
It is a protein that has PPIase activity, and it is presumed that the higher-order structure conversion from opsin to rhodopsin occurs through PPIase activity.

The n1naA gene is expressed in large quantities in the head, which contains many visual cells, and gene fragments that hybridize with the n1naA gene and differ in size have been detected in other parts, suggesting that the n1naA gene is expressed in visual cells. It is thought that it is specifically expressed and functions only in the formation of rhodopsin (Shieh).

B,-H,,Stamnes, M,A 5eav
ello, S,, Harris+G, L, &
Zuker, C,S Nature 338.67-
67-70 (1989)) Schneu, s.
Shortridge, R., Larr
iveee.

D., C., Ono, T., Ozaki, M., & P.
ak, W.L., Proc, Natl.

Acad, Sci USA, 86 (1989).

Based on this example, if we assume that there is a PPIase specifically expressed in T cells in immune system cells, CsA is the T cell-specific PPIase.
It becomes possible to explain that the immunosuppressive effect is exerted by acting on the immune system. This point is in fact supported by the presence of a large number of genes that hybridize with cyclophilin genes in mammalian cells (Ha
endler, B., HoferWarbine
K, R, & Hofer, E, BMBOJ, 6
+947-950 (1987)) However, to date, only one type of cyclophilin has been confirmed to be expressed in all analyzed cells, and there is no diversity in the expressed proteins. Proving that several cyclophilin PPIases exist in one biological species suggests that a specific PPIase works specifically with a specific protein as a substrate. , can be considered logically as the first evidence.

In the example of Rustrum bread mold, where diversity has been reported to exist in a single species, there are two types of cyclophilin mRNAs.
A is known to exist, but these are explained as having different 5' ends resulting from a single gene due to differences in splicing patterns. Among these mRNAs, one encodes cyclophilin present in the cytoplasm, and the other encodes a sequence with an extra signal sequence for translocation to the mitochondria. After the protein enters the mitochondria, it is thought to function as the same molecular species as cyclophilin present in the cytoplasm. Therefore, in this case, there is diversity due to the different localization locations of one type of protein in the cell, that is, the movement of proteins, and there is only one type of active protein (Tropschug, LI N1cho
Ison, DJ, Hartl, F.-U., Hler, H., Pfanner, N., Wac.
Hter, E., & Neupert, W. J., Bi.
ol, Chew, 263.14433-1444
0 (198B) ).

It is known that when a protein is regenerated from a denatured state and returned to an active protein, the isomerization rate of peptide bonds containing proline influences the slow phase of protein reconstitution, that is, it becomes the rate-limiting step. 4 types of proteins,
Concanavalin A, prothrombin, ribonuclease A+% ribonuclease TI, cytochrome C1β-
In an experiment investigating the catalytic effect of PPIase on reconstitution of lactoglobulin, myokinase, chymotrypsinogen, and pepsinogen, it was reported that it was effective on only two types of proteins, ribonuclease T and cytochrome C (Lin.

L-N, + Hasumi+H, & Brandt
S, J, F, Biochis.

Botphys, ^eta 956.256-266
(1988)), this suggests that the types of substrate proteins that one type of PPIase can act on are limited.

Although it is presumed from the above facts that there are several types of PPIases that act on limited types of proteins, their existence has not actually been proven.

Furthermore, there are differences in the types of PPIases present in one biological species, and in particular mammals, which are composed of many cells with extremely diverse functional differentiation, yeast, which is a single cell, and prokaryotes. It is reasonable to think that there are differences in the substrate proteins on which each PPIase acts between E. coli and E. coli, considering the differences in the functions of each cell.

In addition, the hypothesis that the action of CsA is to inhibit PPIase activity has been proposed based on the inhibitory effect of CsA on PP1ase in pigs, but PPIase in other species, such as Escherichia coli, which is very distant from mammals in terms of phylogenetic tree, has been proposed. In Cs
It is not clear whether CsA has an inhibitory effect, and the distribution of CsA binding activity and PPIase
It is not known whether the inhibitory effect of CsA on - is also correlated in lower organisms.
This is important in questioning the pros and cons of the idea that the effect appears by acting on PPIase.

As mentioned above, it has been suggested that PPIase present in various cells may exert substrate-specific effects, and it is expected that the type of PPIase will differ depending on the cell type. It is clear that this has an important meaning when using DNA to produce a specific protein using a specific cell as a host. PPIase that can act effectively
coexist within the host cell. - For boat fishing, the host cell's original proteins include PPIa of the host cell.
Since it is believed that se acts effectively, the use of PPIase and its gene from Escherichia coli, which is often used as a host for the production of useful substances by recombinant DNA technology, for example, is suitable for the above purpose. The presence of cyclophilin in various organisms has been investigated using the binding ability with cyclosporin A as an indicator. Cyclosporin A (Western Kapocha) etc.
binding activity has been detected (Koletsky, A.
J., Harding.

M.J., & Handschumacher, R.J.
, J. Biol, Che+++, 13ヱ.

1054-1059.1986), but no correspondence was established between these activities and PPIase activity.

As mentioned above, although it is clear that PPIase promotes the formation of higher-order structure of proteins,
It was not clear at all whether PIase actually exists and functions, and to what extent it is involved in the formation of higher-order structures of proteins. As one of the means to solve many of the above problems, PP1a from Escherichia coli
It is considered important to isolate se, investigate the properties of PPIase protein, and obtain its gene.

[Problem to be solved by the invention]

As mentioned above, specific examples have been found regarding the involvement of PPIaa in the formation of higher-order structures of proteins, but only one type of PPIase has been identified per biological species, and its utilization in the formation of higher-order structures of proteins has not been confirmed. was limited. Therefore, the present invention provides two types of homogeneously isolated enzymes derived from E. coli, and further provides a method for producing large amounts of the enzymes using genes encoding the enzymes.

[Means to solve the problem]

The above problem is based on the following properties: (1) Acting on the Xaa-P, (Xaa represents any amino acid and pro represents L-proline) bond in the peptide chain to isomerize it. (2) exhibits a single molecular weight of approximately 20,000 daltons in sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis; (3) exhibits a single isoelectric mass of approximately 5.0 in isoelectric focusing; and (4) not inhibited by CsA; a method for producing the enzyme, which comprises collecting the enzyme from cells of Escherichia coli; a gene encoding the enzyme; and this gene. This is achieved by providing a method for producing the enzyme using the method.

The present invention further provides the following properties: (1) Xaa-P in the peptide chain. (xo represents any amino acid and pre represents L-proline) to act on the bond and isomerize it; (2) about 22,000 in sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis (3) exhibits a single isoelectric point of about 9.7 in isoelectric focusing; and (4) is not inhibited by CsA; and Provided is a method for producing the enzyme, which comprises collecting the enzyme from E. coli cells.

To collect PPIase from E. coli, a method commonly used for collecting enzymes from microbial cells can be used. An example of this method is specifically described in Example 1. As the E. coli used as a raw material, any E. coli strain can be used, for example, E. coli ST 24.
9 strains (Kajte, S, +Mtkt+K, +Li
n, E., C.C., & Anraku, Y., F.E.M.
S Microbiol, Lett.

Nagi, 25-29 (1984)) can be used.

The method for measuring the enzymatic activity of PPIase is N. Takah
Ashi et al., Nature, 377, 473-
475 (1989), and this method can also be used for the measurement of E. coli PPIase of the present invention. Specifically, 0.035M HEPF containing 2I11 of 511M2-mercaptoethanol! S buffer pH 7
, 8 into the absorbance meter cell and leave for about 10 minutes until the temperature stabilizes at 25°C. After adding E. coli PPIase, to which 1.68 mM N-SN-5uccinyl
-Ala-Ala-Pro-Phe-50d was added, 3
The 0 reaction is initiated by adding 0.76 mM Chymotrypsin 20d after 0 seconds and is detected by monitoring the absorbance change at 360 n-.

In the present invention, as shown in Example 1, two types of PP
The major component PPIase-β and the minor component PPIase-α were isolated.

These fragments were fragmented by trypsin digestion and cyanogen bromide digestion, and the amino acid sequence of each fragment was determined.The amino acid sequence was further characterized by determining the N-terminal amino acid sequence according to a conventional method. Furthermore, this PPI
An oligonucleotide probe was designed based on the amino acid sequence information of the ase-β protein.

Next, a DNA library derived from E. coli was screened using this oligonucleotide probe to obtain a gene encoding the PPIase of the present invention. The base sequence of this cloned DNA is determined, and the corresponding portion of the amino acid sequence deduced from the base sequence matches the amino acid sequence of the tryptic fragment of the protein (β) isolated from E. coli. From this, it was confirmed that the E. coli-derived DNA encodes large intestine PPIase-β.

Furthermore, this determined amino acid sequence and the known PPIa
From the comparison with the amino acid sequence of se, the PPIas of the present invention
It was confirmed that e-β is a new enzyme different from the already known PPIase.

Furthermore, PPIase-α of the present invention was confirmed to be a novel PPIase by comparing its partial amino acid sequence with the amino acid sequences of known PPIases.

The yeast PPIase-β of the present invention has the amino acid sequence shown in FIG. 7, but is not limited to this.
one or more amino acids in the amino acid sequence shown in the figure are replaced by other amino acids, and/
or one or more amino acids have been added to the amino acid sequence, and/or one or more amino acids have been removed from the amino acid sequence, but still have properties similar to E. coli PPIase-β. Also includes enzymes.

The properties of the enzyme of the invention are described in detail in Examples 2 and 3.

The PPIase of the present invention catalyzes the isomerization of proline peptide bonds in proteins and promotes the formation of higher-order structures of the proteins. can be used to convert it into an active form with Furthermore, the gene encoding the PPIase of the present invention is inserted in an expressible form into a host into which the gene encoding a physiologically active polypeptide has been inserted in an expressible form, and both genes are expressed in the same host. It is expected that this method will enable the direct production of the desired physiologically active polypeptide in its active form. In this case, by providing two different types of enzymes within the same species, it becomes possible to individually utilize different enzymes suitable for activation by promoting the formation of higher-order structures of different proteins within the same species. Furthermore, the PPI of the present invention
Ase does not interact with CsA, so it is useful for research on the mechanism of action of CsA in comparison with PP1ase derived from mammals and yeast, which is inhibited by CsA, and as a reagent for other tests and research.

On the other hand, the gene encoding the PPIase of the present invention is useful for producing the PPIase of the present invention by genetic engineering methods, and by modifying and using the gene, enzyme activity, stability, and intracellular localization can be improved. It is also useful as genetic material for producing PPIase derivatives with altered characteristics, substrate specificity, etc. Furthermore, the gene of the present invention is useful as a probe for screening genes associated with other PPIasePPases.

As described in Examples 5 to 9, when the gene is used to produce PPIase in a microbial host, such as recombinant E. coli, PPIase is produced much more efficiently than when extracted from non-recombinant E. coli. be able to. PPIas produced using recombinants in this way
As shown in Example 10, e has the same properties as PPIase extracted from non-recombinant E. coli.

Next, the present invention will be explained in more detail with reference to Examples.

Escherichia coli (ST 249 strain) cultured under anaerobic conditions with m PPIase 20
0g containing 55M 2-meltocaptoethanol.
1 M Tris-HC4 buffer (pH 7,8), 50
01d, and after repeating freezing and thawing twice, the mixture was stirred using a polytron. To this, 2 g of Norizozyme was added and digested at room temperature for 30 minutes, and further stirred with a Polytron. This was fractionated into a supernatant and a precipitate by centrifugation, ammonium sulfate was added to the supernatant to make it 80% saturated, and the mixture was left overnight at 4°C. Collect the precipitate by centrifugation, and reduce the precipitate to 0.05
% NaN, 10s+M TrisHCj!
After suspending in a buffer (pH 7, 8), dialysis was performed against the same buffer. The dialyzed sample was centrifuged, the precipitate was removed, and the supernatant was transferred to an ion exchange column, DEAE-5 epharos.
e CL-6B column (2,5CIlφX40CI+)
Separated by Peptidylprolyl cis-trans isomerase (PPIase) activity was determined by Takahashi
, N, et al+Nature, 337 (19
89) Measured by the method described in 473-475. Elution of the sample from the column was carried out stepwise by increasing the salt concentration to 0.0.
5M, 0.1M, 0.2M, 0.3M, and 0.5M
and by raising it. The flow velocity at this time is 20
It was id/hr. FIG. 1 shows an elution diagram using the DEAR column. PPrase activity of E. coli was detected in two fractions: a flow-through fraction (salt concentration OM) and a fraction eluted at a salt concentration of 0.1 M. Regarding the total amount of activity, about 10% of the activity was obtained in the flow-through fraction, and about 90% of the activity was obtained in both fractions eluted at a salt concentration of 0.1 M. The component in this pass-through fraction is referred to as a minor component (α), and the component in the fraction eluted at a salt concentration of 0.1M is referred to as a major component (β).

Each fraction was transferred to a 5ephadex-G75 column (
It was further purified by gel filtration using 2,5CIIlφX90CI+). Figure 2a shows the purification process for the minor fraction, and Figure 2A shows the purification process for the main fraction. A lomMTris-HCl (pH 8,0) buffer containing 0.15M NaCl and 0.05% NaNi was used as the eluent, and the flow rate was 10 I/hr. Through this purification step, the main component of PPIase was purified to a single component by sodium dodecyl sulfate (SOS)-polyacrylamide gel electrophoresis and high-performance liquid chromatography using a reversed-phase column. , for minor components, CM-5epharose
Purification was performed using a CL-68 column (1.5 csφ x 20 cm).

CM-5epharose CL-68 column has 10
Equilibrate with a+M sodium acetate buffer (pH 6,0) and elute PPIase with OM NaCl2/30
0111 to 0.25M NaCj! This was carried out using a linear concentration gradient elution method up to 7300 d (Fig. 3).

The flow rate at this time was 12 at/hr. Through this operation, minor components were also purified to a single component using the above purity assay method.

PPIase as the main component is referred to as PPIase-β, and PPIase as the minor component is referred to as PPIas.
It is called e-α.

The molecular weight of PPIase-β obtained in Example 1 was calculated using a 5DS-polyacrylamide concentration gradient gel (12%→3
It was measured by electrophoresis (0% polyacrylamide gel). 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
,000) was used. As shown in Figure 4, approximately 20.00
A single molecular weight of 0 daltons was indicated.

(2)! The isoelectric point of PPIase-β obtained in Ai Rikka Sarashi Example 1 is L
The measurement was carried out by isoelectric focusing according to the method described in the KB Ampholine Isoelectric Focusing Manual. Cytochrome c (prlo, 6) as standard
, whale myoglobin (8,3), horse myoglobin (7,3), pig myoglobin (6,45), pig trifluoroacetyl myoglobin (5,92), azurin (5,65), C-phycocyanin (4,85) ) and C-phycocyanin (4,65), the PPIase-β of the present invention showed a single isoelectric point of about 5.0, as shown in FIG.

(3) ゛ Stay calm - Reversed phase column Aquapore RP-300 (2.1w
mφ×3m: manufactured by Applied Biosystems) in 0.1% trifluoroacetic acid for 45 minutes.
As a result of elution with a 0% acetonitrile concentration gradient at a flow rate of 200 d/min, the P of the present invention was eluted as shown in Figure 6.
PIase-β showed a single peak.

200μ of E. coli PPIase-β was mixed with 50111μ of 0.
1 MNFi, dissolved in HCO, and added 4N T to this solution.
The 0-degradation product hydrolyzed with PCK-conjugated tributrypsin at 37°C for 8 hours was transferred to a reverse phase column 5pher 5RP-1.
8 (2,1111φ×3c+m) and Aquapore
Separation and purification were performed using RP-300 (2.1 mφX3C11) (all manufactured by Applied Biosystems).

In this case, elution was performed using a gradient of O to 100% acetonitrile in 0.1% trifluoroacetic acid or 0.1% pentafluorobutyric acid over 45 min at a flow rate of 200 d/min to separate 18 peptide peaks. Obtained. The results of this elution are shown in FIG.

The amino acid sequences of 7 of these peptide fragments are 47
7A automatic sequencer (Applied Biosystems)
As a result, the following amino acid sequence was obtained.

■ Asn-Phe-Leu-^sp-Tyr-X-A
rg■ Glu-Gly-Phe-Tyr-Asn-A
sn-Thr-11e-Phe-His-Arg ■ Val-11e-Asn-Gly-Phe-Met
-11e-Gln-Gly-Gly-Gly-Phe-
Glu-Pro-Gly-Met-Lys Glu-
Pro-11e-Lys-^5n-Glu-A1a-A
sn-Asn-Gly-Leu-Lys ■ Gly-Thr-Leu-Ala-Met-Ala
-＾rg■ Thr-Gln-Ala-Pro-His
-3er-^1a-Thr-Ala-GlnPhe-P
he-11e-Asn-Val-Val-Asp■ S
sr-Gly-Met-His-Gln-Asp-Va
l-Pro-Lys-GluAsp-Val-11e-
11e-Glu-5er-Val-Thr-Val-5
500g of cyanogen bromide was added to 200N PPIase of Ersin, and the product was decomposed by treating it in 70% formic acid overnight.
8 (2,1mφX3C1l) and Aquapore R
Separation and purification were performed using P-300 (2.1 mφX3C1l) (all manufactured by Applied Biosystems). In this case, elution was performed using a gradient of O to 100% acetonitrile in 0.1% trifluoroacetic acid or 0.1% pentafluorobutyric acid over 45 minutes at a flow rate of 200 i11/min to separate 15 peptide peaks. Obtained. The results of this elution are shown in FIG.

The amino acid sequences of six of these peptide fragments were determined using a 477A automatic sequencer (Applied Biosystems), and the following amino acid sequences were obtained.

■ sial-Thr-Phe-His-Thr-Asn
-Old 5-Gly-Asp-11e-Val-11e ■ l1e-Gln-Gly-Gly-Gly-Phe
-Glu-Pro-GIV■ Lys-Gln-Lys
-Ala-Thr-Lys-Glu-Pro-11e-
Lys-Asn-Glu-Ala-^sn-Asn-G
ly-Leu-Lys-Asn-Thr-Arg-Gl
y-X-Leu ■Ala-Arg-Thr-Gln-Ala-Pro
-His-5er-^1a-ThrAla-Gln-P
he-Phe-11e-Asn-Val-Val-As
p-Asn-Asp-Phe-Leu4-Phe-X-
Gly■ ^5p-Glu-Val-Asp-Lys-
11e-Lys-Gly-Val-^1aThr-Gl
y-＾rg-Ser-Gly@H45-Gln-A
sp-Val-Pro-Lys-Glu-Asp-Va
l-11e1e (5) N-amino 1 The sequence of the N-amino acid terminal of PP1ase-β is 477A
Amino acid sequencer (Applied Biosystems)
The following sequence was obtained.

Mer-Va 1-Thr-Phe-Hi 5-Thr
-Asn-His-Gly-Asp-11e-Val
-I 1e-Lys-Thr-Phe-Asp-Asp
-Lys-^1a-Pro-Glu-Thr-Val-
Lys-Asn-Phe-^5p-Tyr (6) 0.5 id of 6 NM to 5N purified PPIase-β
CI was added, the tube was degassed, the tube was sealed, and the tube was hydrolyzed at 110° C. for 24 hours. After drying this hydrolyzate under reduced pressure, the amino acid composition was measured using an amino acid analyzer C-300 (JEOL Ltd.). The results are shown in Table 1.

】-"-Table 1 (1) The numbers in parentheses indicate the number of amino acids obtained from the determined amino acid sequence.

(2) There are two Val-Val bonds in the amino acid sequence,
There are three Valle bonds.

Cs on the activity of purified E. coli PPIase-β
The inhibitory effect of A, N, Takahashi et al., Na
true.

377, 473-475 (1989). The results are shown in FIG. This figure shows a comparison of the degree of inhibition of E. coli PPIase and porcine PPIase by cyclosporine A. At a concentration of cyclosporin A at which porcine PPIase and yeast PPIase are almost completely inhibited, E. coli PPIase β is
porcine PPIase and yeast PPI are rarely inhibited
In the case of CsA, these activities are inhibited by the binding of CsA, so it is thought that there is a positive correlation between the binding activity of CsA to PPIase and the inhibitory effect of CsA on PPIase activity. Since the binding activity of CsA to PPIase and the inhibitory effect of CsA on PPIase activity are directly proportional, the effects of CsA on various organisms, such as antifungal, antischistosoma, and antimalarial effects, are due to the effects of CsA on PPIase. It is presumed that this occurs through the inhibitory effect on binding activity, ie, PPIase activity. On the other hand, Cs to E. coli PPIase
The fact that almost no inhibitory effect by A is observed is also consistent with the fact that although CsA binding activity exists in all eukaryotes examined, it has not been detected in E. coli.

X-114PPIase” of ゛(1) minute 3”b
Similar to the experiment described in Example 2 (1), Escherichia coli PPIa
The results of measuring the molecular weight of se-α are shown in FIG. 4, and the single molecular weight determined from this figure was 22,000.

(2) E. coli PPIa at the same time as the experiment described in Example 2 (2)
The results of measuring the isoelectric point of se-α are shown in FIG. The single isoelectric point determined from this figure was p19.7.

(3) Calm analysis showed a single peak in the same method as described in Example 1 (3). The results are shown in Figure 6. Under this separation condition, PPIase-α becomes PPIase
- It eluted about 3 minutes earlier than β.

Seventeen peptide peaks were obtained in the same manner as PPrase-β. Among them, for 5 peptide fragments, 4
As a result of determining the amino acid sequence using a 77A automatic sequencer, the following amino acid sequence was obtained.

■Ala-Pro-Val-Ser-Val-Gin
-Asn-Phe-Val-AspTyr-Val-A
sn-5er-Gly-Phe-Tyr-^5h-Ac
n-Sr■ Thr-8la-^5p-Lys-^5
p-5er-X-Ala-Asp-Gln-Phe-P
he-11e-Asn-Val-^1a-^sp-^5
n-Ala■ X-Met-Asp-Val-Ala-
^5p-Lys-11e-5er-Gln-Val-P
r.

■ Val-11e-Pro-Gly-Phe-Met
-11e-Gln-Gly-Gly-Gly-Phe-
Thr-Glu ■ Asp-Phe-Gly-Tyr-Ala-Val
Ten peptide peaks were obtained in the same manner as the amino PPIase-β of -Phe-Gly-Lys cyanide. The amino acid sequences of four of the peptide fragments were determined, and the results are shown below.

■ l1e-Gln-Gly-Gly-Gly-Phe
-Thr-Glu-Gln-(Met)■Ala-A
rg-Thr-Ala-Asp-Lys-Asp-5e
r-X-Ala■ Asp-Val-^1a-Asp-
Lys-11e-Ser-Gln-Val-Pro-X
-His-Asp-Val-Gly ■ Gln-Gln-Lys-Lys-Pro-Asn
-Pro-Pro-11e-Lys-Asn-Glu-
Ala-Asp-Asn-Gly-Leu-Arg-A
sn-X-Arg-Gly (5) N-r amino PPIase in the same manner as described in Example 2(5)
The N-terminal amino acid sequence of -α was determined and the following results were obtained.

A la-Lys-Gly-Asp-Pro-His-
Val-Leu-Leu-Thr-Thr-A 1a-
G 1y-Va 1- Asn-Ile-G 1u
-Leu-X-Leu-Asp-Lys -Xys (6) PPIase in the same manner as described in Example 2 (6)
The amino acid composition of -α was determined. The results are shown in Table 1.

Biol, 11.476 (1965)) was used to prepare high molecular weight DNA. Approximately 24% of this high molecular weight DNA was added to a solution of 20 pl [10 mM Tris-H (J (p
H7,5), 100a+M NaC7! ,6tsM M
gC1z, 6aM mercaptoethanol, 0.1% gelatin, 10 units of Bgl II (Nippon Gene) and 10 units of former ndllI (Nippon Gene)]
After digestion for 3 hours at 37°C, electrophoresis was performed on a 0.8% agarose gel using the Southern method (S
out thern.

E, M. (1975) J. Mo1. Biol, 9
8.503) according to the membrane filter (Ame
rsha- Inc., Hybond-N).

The DNA was fixed on the filter by UV irradiation for 2 minutes, and hybridization between the probe and the DNA on the filter was performed according to the following method. The probe was made using an automatic DNA synthesizer (Applied Biosystems) based on the amino acid sequence from 59th methionine to 67th proline of E. coli PPIase-β.
5'ATGAA (model 380B)
AG) CA (AG) AA (AG) GC (TCAG) AC
A DNA strand named CAAAGAACC-3' was selected. Synthetic NA (20ps+oles) at 50mM T
ris-H(J (pH 7,5), 10+wM
gCl2t, 5IIIM dithiothreitol, 100
μCi (γ-”P)ATP (3000Ci/mm
ol, Amershas+) and 12 units of T
4 Solution containing polynucleotide kinase (Takara Shuzo Co., Ltd.) 5
5 by reacting in 0111 at 37°C for 60 minutes.
The 'end was phosphorylated and labeled. Add the above filter to the prehybridization solution [5x Denhardt's solution (100x
Denhardt's solution = 2% bovine serum albumin, 2% Ficoll, 2% polyvinylpyrrolidone), IM NaCl
, 50a+M Tris-H (J! (p) 17.5
), 10s+M EDT^ (pH 8,0), 0.1
% sodium dodecyl sarcosinate and 20 cpm/d of sonicated salmon sperm DNA) for 1 hour at 37°C.
(containing solution) for 15 hours at 37°C. This filter is 6XSSC (20XSSC = 3M NaCj!
X-ray film (Kodak, χAR- 5) was exposed to light at -80°C for 4 days.

As a result of development, Bgl II/Hin with a length of approximately 1 kb
Since it became clear that the d m DNA fragment hybridizes with the probe, we decided to create an E. coli gene library containing approximately 1 kb DNA fragments as follows.

Approximately 50x of the above E. coli high molecular weight DNA was added to the solution (10a+M
Tris-HCj' (pH 7,5), 100mM
NaCl, 6mM MgCfz, 6+wM mercaptoethanol, 0.1% gelatin, 200 units Bgl
Nippon Gene Co., Ltd.) and 200 units of the former NDL
After digestion in I [Nippon Gene Co., Ltd.] for 3 hours at 37°C,
．． Electrophoresis was performed on an 8% agarose gel, and DNA fragments with a length of about 1 kb were collected using the glass powder method (Bio-101
Separated and purified by Gene C1ean, Inc.).

Recovered Bgl II/Hind m DNA
About 50 ng of the fragment was reacted with about 1100 n of pUc119 vector digested with BamHI and old ndl in Takara Shuzo's DNA ligation kit A solution 40 m and B solution 8 I at 16°C for 15 hours to ligate both DNAs. A replacement plasmid was obtained. This reaction solution 20J1
1, competent cell JM 109 strain (Epicuri
an coli” 5TRATAGENE) 400J
11 was transformed according to the manufacturer's recommended method.

The transformants were grown on an X-Ga1 plate with a diameter of 90 m [50
■/M15-bromo 4-chloro-3-indolyl-β
-D-galactopyranoside, 80 tar/dl isopropyl-β-D-thiogalactopyranoside, 254/d ampicillin, LB medium (1% Batato tryptone, 1% N
aCl1, 0.5% yeast extract) and 1.5% agar]. 3 of these plates
As a result of overnight culturing at 7°C, about 1500 transformants were obtained as white colonies of E. coli. With this, E. coli PP
It was used as a library for 1aseβ gene screening.

Filter recombinant colonies (Hybond-N.

Amersham) and diluted with 0.5 N NaOH1
, 3MM filter paper soaked in 5M Na (J!
LM Tris-HCI (pH 7) for 5 minutes with the colony-attached side facing up.
, 5) on the same filter paper soaked in 1.5 M NaCl1.
I left it for a minute. Furthermore, the filter was washed with 2xssc solution, air-dried, and then UV irradiated to immobilize E. coli DNA on the filter. The filter thus obtained was synthesized under the same conditions as described above, and after hybridization was performed using NA as a probe, the filter was washed, exposed to X-ray film, and developed. The nine positive clones obtained were used for subsequent analysis. That is, each desired colony on the plate was inoculated into LB medium containing 25 R/dl of ampicillin, cultured with shaking at 37°C overnight, and 1.5 d of the resulting culture was subjected to alkaline lysis ( Ish-Horowicz, D., & J.F.

Burke (1981) Nucleic Ac1d
s Res, 9.2989).
NA was prepared. DNA of nine clones thus obtained
After double digestion with EcoRI and old nd■, 1% agarose electrophoresis was performed, and the same Sou
I did a thern analysis.

As a result, only one clone containing a 950 bp E. coli DNA fragment was a positive clone. This plasmid DNA was named pEPPIb.

This pEPPIb was integrated into M13p-based phage DNA, and the nucleotide sequence was determined by the dideoxy method (Figure 10). The isolated gene encodes E. coli PPIase-β since the amino acid sequence deduced from the nucleotide sequence completely matches the amino acid sequence of the peptide obtained from the main component of E. coli (PPIa!0-β). It turned out to be genetic. This gene encodes a protein consisting of 1644 amino acids, and the molecular weight calculated from the amino acid sequence is 18.1
84, which is in good agreement with the molecular weight estimated from SDS electrophoresis. The amino acid composition also matched well with the measured values (Table 1).

FIG. 20 shows a comparison of the entire amino acid sequence of E. coli PPIase-β and the partial amino acid sequence of PPIase-α with the sequences of other cyclophilins.

In this diagram, amino acids that are conserved at homologous positions in all species, including E. coli, are boxed. The amino acid sequence of E. coli PPIase-β shows about 25% homology when compared with cyclophilin/PPIase derived from other species, and in particular, about 30-30% homology is observed from the amino terminal side.
It can be seen that regions of high homology are found between up to 70 residues and between about 100-120 residues. In addition, in the case of E. coli PP1ase-α, amino acid residues in regions that are highly homologous to PPIase-β and highly homologous to sequences of other species are conserved, albeit partially. You can see that. Therefore, both E. coli-derived PPIase-β and α are cyclophilin-like PPIases.
It is thought to be PIase. However, both PPIases from E. coli differ from cyclophilin/PPIases from other species, in this case eukaryotic species, and CsA
By comparing these structures, it is possible to determine the CsA binding mode of other species, P
It is believed that this provides the basis for analysis of the action mechanism of PIase as an enzyme.

In the case of porcine PPIase, the SH modification reagent causes modification of the four cysteines and inactivates the enzyme activity, but binding to CsA prevents modification of one of the cysteines. . CsA protects cysteine from modification by SH reagents by directly interacting with cysteine residues as active groups, since dissociation by dilution of CsA after modification restores its enzymatic activity. It is believed that

(Fisher.

G., Wittsann-Liebold, B.
,, Lang+ l Kekfhaber.

T., & Schmid, F.J., Nature.
33? , 476-478 (1989)) However, not only is the enzymatic activity of both E. coli-derived PPIases not affected by CsA, but SH
Its activity is not affected by modifying reagents.

From this point of view, when comparing the amino acid sequences in FIG. 20, there is not a single cysteine residue that is completely conserved among PPIases derived from all biological species.

It has been shown that it binds CsA and inhibits its enzymatic activity. Pig PPIase and yeast PPIas
e (in the previously patented yeast PPIase related documents), and is not affected by CsA. E. coli PPIase-
Comparison of patterns of β humanobacy (Kyte, J, M
, &Doolittle, R.F., J., Mo1
．． Biol, E., 105-132 (1982)
) On the diagram (Figure 21), look at the position of the cysteine residue that is common to CsA-sensitive pig and yeast PPIases and does not exist in E. coli. Pig Cys-115 and yeast Cys-
It can be seen that 117 is located at a very modest position on the humanopathic pattern.

However, there is no cysteine residue in the similar position in E. coli PPIase-β. From the above, pig Cy
s-115 and yeast Cys-117 are presumed to be cysteine residues that directly interact with CsA. but,
Based on the example of E. coli, there remains some doubt as to whether this cysteine residue is an active group directly involved in the expression of enzymatic activity. At the very least, in the case of E. coli, there is no evidence that the cysteine residue is an active group; rather, two regions that were found to be highly homologous in all species, obtained by comparing amino acid sequences, are responsible for active expression. considered to be important.

In addition, E. coli ε5cheri containing the above plasmid
rhia coli HBIOI/pEPPIb was submitted to the Institute of Microbiological Technology, Agency of Industrial Science and Technology, No. 1104.
No. 2 (FERM P-11042).

1 boat 1. - Boothmi BLEPPIb (1st
Figure 1) Plasmid containing E. coli PPIaseβ gene (
pEPPIb)6R in 100ml solution (50mM Tr
is-HCJ (pH 7,6), 7 mM MgCl
z, 1501++M KIJ! , Pvu in 2Q units
50 tons after extinguishing for 180 minutes at 37°C in Nippon Gene Co., Ltd. l of phenol/chloroform mixture (
The extraction using 1:1) was repeated twice to remove proteins, and the DNA recovered by ethanol precipitation was dried using a centrifugal evaporator. The obtained DNA was added to 100111 mung bean nuclease buffer (Takara Shuzo DNA d
eletion kit), add 2111 mung bean nuclease (Takara Shuzo Co., Ltd. kit),
After 60 minutes of reaction at 37°C, 50d of phenol/chloro-
Extraction with a form mixture (1:1) was repeated twice to remove proteins, and the DNA was recovered by ethanol precipitation and dried using a centrifugal evaporator. Add this DNA to solution 30
d (100s?l Tris-HCl1
(pH7,6), 7m? I MgCj! *, 5
0m? tNaCj! , 2Q unit EC0RI (
1.2% after reaction for 60 minutes at 37°C
Agarose gel electrophoresis was performed, and the inserted DNA fragment was analyzed using the glass powder method (Gene C1ean, Blo-1
01 company) and 1011! TE solution (10 m
M Tris-HCI (pH 7,5), 1sMED
Extracted with TA).

The obtained inserted DNA fragment 4 was digested with both EcoRI (Ppon Gene) and EcoRV (Bluesc) restriction enzymes.
About 15 ng of riptII SK+) was added to 30 m of Takara Shuzo DNA Ligation Kit A solution and 5.
5 d of the same solution B and reacted at 16°C for 16 hours to obtain a recombinant plasmid in which both were linked.

Calcium chloride method (Man
del, M. & Higa, A. (1970) J.
, Mo1. Biol.

Escherichia coli XL4 Blue was transformed using the method (Koh, 159). Transformants were grown on X-Ga1 plates (155
g/d5-bromo-4-chloro-3-indolyl-β-
D-galactopyranoside, 8011 g/Id isopropyl β-D-thiogalactopyranoside, 50 n/d ampicillin, 1% NaCj! , 0.5% yeast extract, 1%
Tryptone, 1.5% agar) was selected as a white colony obtained by culturing at 37°C for 16 hours.

The transformants were grown in LB medium (1% Macl, 0.5% yeast extract, 1%
tryptone) and cultured at 37°C for 6 hours. From this culture solution, the alkaline lysis method (Birnboigi, H.
C.

l Doly, J. (1979) Nucleic
Plasmid DNA was prepared using Aclds Res, 7.1513) and dissolved in 30I11 TE solution. 1/3 of the obtained DNA was added to a solution (50mM Tr
is-HCj! (pH 7,6), 7sM
MgCj! z, 100mM NaCl,25
mg/, J ribonuclease A, 5 units of Xho I
(Takara Shuzosha), 5 units of Ram! (I (Nilapon Gene)) for 60 minutes at 37°C, 0.8% agarose gel electrophoresis was performed to confirm the transformant having a plasmid containing the desired inserted DNA fragment. 200d L containing 50g/d ampicillin
The cells were cultured in medium B, and plasmid DNA was prepared by the alkaline lysis method. The obtained plasmid DNA was subjected to cesium chloride density gradient centrifugation (Maniatis, T. et al.
19B2) Molecular Cloning;A
Laboratory Manual, Col.
Spring Harbor Laboratory)
It was further purified by This plasmid was converted into PBLEPPI
Named b.

111 Example J1 Boothmi BLΔEPPIb
(Figure 12) pBLI of 29N! PPIb was added to the solution 1 d (50n+
M Tris・HCj! (pH 7,6), 7mM
IIlgCjh, 150mM NaCj! ,
After digestion in 200 units of 5ail at 37°C for 12 hours, the restriction enzyme was inactivated by incubation at 5°C for 15 minutes. Add 10111 of 1mM Th to 500I of this solution.
io-dNTP (Stratagene, EXO/M
UNG deletion kit) and 10Ill
Add Klenoev fragment (Takara Shuzo), 37
The cut site was smoothed by reacting at °C for 45 minutes. this DNA
The solution was extracted with a phenol/chloroform mixture (1:1) to remove proteins, and then DNA was recovered by ethanol precipitation. Furthermore, add this DNA to 25Ill of solution (50+
eMTris-HCj! (pH 7,6), 7mM
MgCl1z, 60mM NaCl, 40 units of Bi
After protein removal, ethanol precipitation was performed and drying was performed using a centrifugal evaporator.

The obtained DNA was dissolved in 1001J1 Exom buffer (Takara Shuzo DNA deletion kit),
After adding IJll's exonuclease ■ (same kit from the same company), the reaction was carried out at 23°C, 10 m was sampled every 20 seconds, and 10 I mung bean nuclease buffer was added and transferred to water to stop the reaction. The 10 samples thus obtained were mixed and incubated at 65°C for 5 minutes to inactivate exonuclease ■.
111 mung bean nuclease (same kit from the company)
was added and reacted at 37°C for 60 minutes to perform deletion. After protein removal, DNA was collected by ethanol precipitation, and 1/4 of the DNA was added to 100111 total sake brewing DNA Ligat.
16° in ionkit A solution and 12Ill of the same B solution
The DNA was circularized by reacting for 120 minutes.

In order to remove unreacted plasmid DNA from the above deletion reaction, the above circularized DNA was digested with 5all. E. coli XL-I Blue was transformed using the obtained plasmid by the calcium chloride method. Plasmid DNA was extracted from the obtained transformant by the same method as described above.
After double digestion with Xho I (Takara Shuzo Co., Ltd.) and EcoRI (Ppon Gene Co., Ltd.), 1.2% agarose gel electrophoresis was performed to obtain the inserted DNA of the desired length.
Transformants carrying the fragment were selected. The base sequence near the 5' end of the PPIase-β gene in the plasmid DNA molecules of these transformants was determined using TOYOBO's M13
Determined using Sequencing Kit, and the following P
One clone considered to be most suitable for constructing a P1ase-β gene expression plasmid was selected, and this plasmid was named pBLΔEPPIb.

1 Boosumi ^Ttr EPPIb pBLΔEPPlb plasmid DNA IOn obtained in Example 6 was mixed with 50 Ill of a solution (50mM Ttr).
ris-HCl (pH 7,6), 7a+M
MgCj! z, 100mM NaCl,2
0 units of Xho I (Takara Shuzo)] at 37°C and 120°C.
After a minute reaction, protein removal treatment was performed, and then DNA was recovered by ethanol precipitation. This DNA is 50111 Kl
eno-buffer [7sMTris-HCj! (pH7
,5), 7sM MgCl1z, 100mM Mac
l, 0.1 mM EDTA, 5 units of Kleno
-Fragment (Takara Shuzo Co., Ltd.)] for 50 minutes at 37°C to blunt the Xho I cleavage site, followed by protein removal treatment, and then ethanol precipitation to recover the DNA.

The DNA thus obtained was further added to a solution of 30 m (50 m
MTris-HCj! (pH7,6), 7s+H
MgCl1z, 100sM10Os, 20 units of Ba5
After reaction for 60 minutes at 37°C in HI (-'-7 Bongene), the DNA was subjected to 1.2% agarose gel electrophoresis, and the inserted DNA was recovered by the glass powder method and extracted with 10JTE solution.

On the other hand, PPL-TNF (Ikenara 3+Che
m, Pharm.

trp promoter tr on Bulletin in print)
Vector pA obtained by ligating the heavy digest of Pst I /C1a containing the p LSD sequence with the larger fragment generated by double digestion of pAT153 with the same enzyme.
First, 10 g of DNA of Ttrp (described in Japanese Patent Application No. 63-37452) was added to C1a in the same manner as above.
After digestion with I (Bio-Rad), the cut portion was digested with Klen.
Blunted with oi4 fragment and further Baa+HI
After digestion, the inserted DNA was separated by 1.2% agarose gel electrophoresis, recovered by the glass powder method, and extracted with 10JTE solution.

The thus obtained D containing the E. coli PPIaseβ gene
pATtrp 4 jll opened with NA fragment 2j11
30m of Takara Shuzosha DNA Ligation kit
Mix A solution and 6 ml of the same B solution and incubate at 16°C for 16 hours.
After a period of reaction, a circular recombinant plasmid in which the two were linked was obtained. This expression plasmid was transformed into pATtrp EPPI
Named b. The resulting expression plasmid pATtrp
Escherichia coli 1 was prepared using EPPIb using the calcium chloride method.
(The 8101 strain was transformed.

Using this transformant, the following Escherichia coli PPIaseβ
We attempted an expression experiment in E. coli. In the following experiments, a transformant containing the pAT trp plasmid was used as a control. Transformant glycerin stock 501
No. 11 was inoculated into LB medium 5II& containing 50 g/llft ampicillin, and cultured overnight at 37°C.

50ml of this preculture solution was mixed with 100d of M9CA-aaip medium containing 50μ/d of ampicillin (0.05% NaC).
A, 0.6% Na, HPO, 0.3% KH! P.O.,,
0.1%NH4Cj! , 0.2% Casamino Acids, 2mM
MgSO4, 0,2% glucose, O,l mM C
aCl z , 50g/Id thiancillin, ptt7
．． 4) and cultured with shaking at 37°C.

When the turbidity (absorbance at 600 Mm) reached approximately 0.3, 3-β-
After indole acrylic acid (IAA) was added to induce expression, the culture was further cultured with shaking at 37°C for about 20 hours. It was found by densitometry analysis of the gel after electrophoresis that the amount of PPIase-β produced by the transformant was more than 50 times greater than that of the control (FIG. 13). It can be seen that PPIaseβ is expressed in large amounts in the soluble fraction of the transformants.

LB-A-medium (10 g/l bactodribton,
5g/! Yeast extract, 5g/1Nacj! ,50x/d
Glycerol stock 50111 of the above transformant was inoculated into ampicillin) and cultured overnight at 37°C.

0.5 of this culture solution was inoculated into 50 Jd of LB-Asp medium and cultured again overnight. Spread this culture solution 4af for 400 m
di inoculated into 9CA-AIip medium and incubated at 37°C for 2 days.
After culturing for 1 hour, 0.81 d of 20 μ/d3-β-indoleacrylic acid (IAA) solution was added, and the mixture was further cultured for 24 hours.

This 400 d culture was carried out in 10 batches, and about 41 culture fluids obtained were collected and centrifuged at 4°C for 5 minutes at 6000 rpm to obtain 11.8 g of wet bacterial cells.

11.8 g of this bacterial body was mixed with 0.1 M Tris-HIJ (9H) containing 5 tsM 2-mercaptoethanol.
1,5) The suspension was suspended in 50Ii and 11.8μ of egg white lysozyme was added and treated at 30°C for 1 hour. To further complete cell disruption, 1B was treated with ultrasound for 1 minute.
Centrifugation was performed for 30 minutes using a 000 Or, p, and Peng, and the supernatant was collected. This supernatant was mixed with 101 containing 0.05% NaN+.
DEAE-5ep equilibrated with the same buffer after dialysis 3 times against gM Tris-HC1 pH 8,0,31
harose CL-6B (2,5C1φX40C1
1) Separation was performed using a column. The enzyme activity of peptidylprolyl cis-trans isomerase is determined by TakahaShi,
N, et al. Nature 337.473-475 (1
989). Elution of the protein from the column was carried out using salt concentrations of 0.05M, 0.1M, and 0.2M.
, by increasing it stepwise to 0.3M,
The target enzyme was detected in the eluate with a saline concentration of 0.1 M (Figure 14).

Fractions 176-196 containing enzymatic activity were concentrated with 80% saturated ammonium sulfate and treated with 0.15M Mace and 0.05%
105M Tris-HCj containing NaN3! pH8
,025ephadex-G75 column (2,5CIl
φX90C11) to obtain a purified sample (Fig. 15). Fractions 64-78 in this figure were collected and used as purified fractions, and the enzyme was characterized as follows.

M E PPIase-
Comparison of constant molecular weights was determined by sodium dodecyl sulfate-polyacrylamide concentration gradient gel (12%→30% polyacrylamide gel) electrophoresis.

Phosphorylase b (molecular weight 94.00
0), bovine serum albumin (67,000), ovalbumin (43,000), carponic anhydrase (30,000), soybean trypsin inhibitor (20,100) and α-lactalbumin (14,4
00) was used. As shown in Figure 16, molecular weight 20.0
It exhibited a single molecular weight of 0.00 daltons and was indistinguishable from the native enzyme.

(2) Comparison of the isoelectric points of Tosori Tatsuhi was measured by isoelectric focusing according to the method described in LKB's Ampholine Isoelectric Focusing Manual. As standards, cytochrome C (pi 10.6), whale myoglobin (8,3)
, horse myoglobin (7,3L pig myoglobin (6,
45), porcine trifluoroacetyl myoglobin (5,
92), azurin (5,65), and C-phycocyanin (4,85, 4,65) showed a single isoelectric point of about 6.2, as shown in Figure 17. It could not be distinguished from the natural enzyme.

(3)' Reversed phase column Aquapore RP-300 (2,1a
aφ x 3 cm; manufactured by Applied Biosystems) in 0.1% trifluoroacetic acid for 45 minutes.
200111/ with an acetonitrile concentration gradient of oo%
It eluted at a flow rate of minutes. The purified enzyme showed a single peak, and the elution position could not be distinguished from that of the native enzyme.

Furthermore, an attempt was made to mix and separate the recombinant type and the natural type, but a single peak was obtained, and no difference was found in their behavior on a reversed phase column (Figure 18).

(4) Amino rl The PPIase preparation obtained in Example 3 was used as model 4.
When analyzed using a 77A protein automatic sequencer (Abride Biosystems), the sequence was completely identical to the natural enzyme sequence up to 20 residues starting from the amino terminal methionine as shown below.

0 11e-Val-11e-Lys-Thr-Phe-^
5p-Asp-Lys-Ala, -0(5) 7.1m 0.5d of 6NH (J) was added to 20g of purified PPIase, degassed, sealed, and hydrolyzed at 110'C for 24 hours. .

After drying this hydrolyzate under reduced pressure, the amino acid composition was measured using an amino acid analyzer model JLC-300 (manufactured by JEOL Ltd.). The results are shown in Table 1. This table shows the composition values for the recombinant type and the natural type, and good agreement has been obtained.

(6) Mouse millet ■ 1 pair of plate-shaped The specific activities of the recombinant and natural enzymes are N.

Takahashi et al. Nature 377, 473
-475 (1989), the activity was measured based on the value.

As a result, the specific activity of the natural enzyme was 51,480 arbitrary units/A!
,. IUnit, whereas recombinant stopping activity 528
It was 76 arbitrary units/A28°1 Unit, and could not be distinguished in terms of specific activity. (Figure 19).

[Brief explanation of drawings]

Figure 1 shows the E. coli extract as DEAE-5 epharos.
e The state of elution when separated using a CL-6B column is shown. In Figure 2, (a) represents the minor fraction shown in Figure 1 (
The figure shows the elution when the α-fraction) was purified by gel filtration using a 5ephadex-G75 column. In the figure, -- indicates the elution of the protein detected by absorbance at 280 nm, and -〇- indicates the elution of the PPIase activity. shows. (b) shows the results for the main fraction (β fraction) in FIG. Figure 3 shows PPIas separated according to Figure 2(a).
e-cr fraction to CM-5epharose CL-6
The state of elution when further purified using a column is shown. Figure 4 shows purified PPIase-a and PPIa.
The results of measuring the molecular weight of seβ are shown. Figure 5 shows purified PPIase-a and PPIas.
The results of measuring the isoelectric point of e-β are shown. In FIG. 6, (a) shows that purified PPIase α shows a single peak in the reverse phase column Aquapore RP-300, and (b) shows that PPIase-β
The results are shown below. In Fig. 7, (a) shows the peptide fragments generated by trypsin decomposition of PPIase-α,
Shows the results of separation by 5RP-18, (b) shows P
Similar results are shown for PIase-β. In Figure 8, (a) shows the peptide fragments generated by cyanogen bromide decomposition of PPIase-α,
The results of separation by ore RP-300 are shown, (b
) shows similar results for PPIase-β. FIG. 9 is a graph showing the influence of CsA on the activity of E. coli PPIase of the present invention in comparison with the activity of porcine PPIase, and shows the relationship between the CsA concentration and the residual activity of PPIase. Figures 10-1 and 10-2 show PPIase of the present invention.
The nucleotide sequence and deduced amino acid sequence of the E. coli-derived gene encoding β are shown. FIG. 11 shows the process of construction of plasmid pBLΔEPPIb. In FIG. 12, (A) is the expression plasmid pATtr
The process of constructing pEPPlb is shown, and [B] shows the base sequence of the connecting region between the trpE promoter and the PPIaseβ gene in the plasmid pATtrp EPPlb. FIG. 13 shows the state of large-scale expression of PPIase-β by transformed E. coli. Figure 14 shows PPIase produced by recombinant E. coli.
The state of elution when β is separated using a DEAE-5 epharose CL-68 column is shown. Figure 15 shows the 5e fraction obtained by the elution shown in Figure 14.
The state of elution when purified by gel filtration using a phadex-G75 column is shown. Figure 16 shows natural PPIase-β and recombinant PPIas.
The results are shown when comparing e (β) in 5DS-electrophoresis. Figure 17 shows natural PPIase-β and recombinant PPIas.
The results of comparing the isoelectric points with e (β) by isoelectric focusing method are shown. Figure 18 shows natural PPIase-β and recombinant PPIa.
se (β) and reverse phase column Aquapore RP-3
00 is shown. Figure 19 shows natural PPIase-a, natural PPI
Figure 2 shows the results of comparing the specific activities of Ase(β) and recombinant PP1ase-β. FIG. 20 shows a comparison of the amino acid sequences of PPIase or cyclophilin from various sources. FIG. 21 shows the humanobacy patterns of the amino acid sequences of PPIases from various sources.

Claims (1)

[Claims] 1. The following properties: (1) X_a_a-P_r_o(X_a
_a represents any amino acid, and P_r_o is L
(2) exhibits a single molecular weight of approximately 20,000 Daltons in sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis; (3) isoelectric An Escherichia coli peptidyl prolyl-cis-trans isomerase β exhibiting a single isoelectric point of about 5.0 in point electrophoresis; and (4) not being inhibited by cyclosporin A. 2. The enzyme β according to claim 1, which has the following amino acid sequence: [There is an amino acid sequence]. 3. A method for producing Escherichia coli peptidyl prolyl-cis-trans isomerase β, which comprises isolating the enzyme according to claim 1 or 2 from Escherichia coli cells. 4. A gene encoding Escherichia coli peptidyl prolyl-cis-trans isomerase β according to claim 1 or 2. 5. The gene according to claim 4, which has the following base sequence: [There is a base sequence]. 6. An expression plasmid containing the gene according to claim 4 or 5. 7. Escherichia coli peptidyl prolyl-cis trans isomerase β is collected from the culture by culturing a host microorganism transformed with the expression plasmid according to claim 6.
Method for producing trans isomerase β. 8. The following properties: (1) X_a_a-P_r_o(X_a
_a represents any amino acid, and P_r_o is L
(2) exhibits a single molecular weight of approximately 22,000 Daltons in sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis; (3) isoelectric An Escherichia coli peptidyl prolyl-cis-trans isomerase alpha which exhibits a single isoelectric point of about 9.7 in point electrophoresis; and (4) is not inhibited by cyclosporin A. 9. The Escherichia coli peptidyl prolyl-cis-trans isomerase α according to claim 8, which has the following partial amino acid sequence: [There is an amino acid sequence]. 10. A method for producing Escherichia coli peptidyl prolyl-cis-trans isomerase α, which comprises isolating the enzyme according to claim 8 from Escherichia coli cells.