JP2000083673A - Fkbp type ppiase derived from thermophilic archaebacteria belonging to aeropyrum pernix and gene encoding the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject new gene, encoding a protein having a specific amino acid sequence and peptidyl prolyl cis-trans-isomerase activity and usable e.g. for production of the above enzyme useful e.g. for regeneration of denatured proteins and stabilization of protein reagents. SOLUTION: This new gene is such one as to encode a protein having an amino acid sequence described in the formula or a protein having an amino acid sequence wherein one or plural amino acid(s) is (are) deleted, substituted or added in the amino acid sequence described in the formula and consisting of a protein having peptidyl prolyl cis-trans isomerase activity and to have high heat resistance and to be useful e.g. for regeneration of denatured proteins, stabilization of protein reagents, production of recombinant proteins and elucidation of new immunosuppressive agents and physiologically active substances, and usable e.g. for production of the above enzymes. This gene is obtained by separating genomic DNA of aeropyrum pernix K1 strain, a thermophilic archaebacterium and conducting PCR of the DNA by using a primer consisting of a partial sequence.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to an enzyme having high heat resistance and useful for regeneration of denatured proteins, stabilization of protein reagents, production of recombinant proteins, and search for novel immunosuppressants and bioactive substances. Peptidyl prolyl cis-trans-isomerase
isomerase (hereinafter referred to as PPIase) and a gene encoding the same.

[0002]

2. Description of the Related Art Proteins are polypeptides in which a plurality of amino acids are linked by peptide bonds. In order for a protein to exhibit its properties, a specific tertiary structure (steric structure) formed by intramolecular or intermolecular interaction is important. In general, when an environmental stress such as heat is applied to a protein, its three-dimensional structure changes, and its characteristics often disappear irreversibly. Therefore, how to keep a protein in a stable state against such environmental changes has always been cited as an issue in handling proteins. In addition, in production of useful proteins by genetic recombination techniques, there is a problem that a protein overproduced in a host bacterium such as Escherichia coli produces a target protein as an inactive inclusion body, thereby lowering production efficiency.

[0003] As a solution to the above-mentioned problems, intensive studies have been made so far, and various improvement plans have been proposed.
In recent years, interest has been increasing in molecular chaperones as factors involved in the formation and structural changes of proteins. Molecular chaperones are a group of heat shock proteins that are produced when cells are exposed to various environmental stresses such as temperature changes. These are widely present in both prokaryotes and eukaryotes, and GroE is particularly well known as a molecular chaperone produced from Escherichia coli. This GroE has been shown to be nonspecifically involved in the formation of three-dimensional structures of proteins, regardless of the type of protein. For example, Gr
GroEL, which is a component of oE, has a characteristic structure composed of a total of 14 subunits, in which a donut-shaped structure in which seven subunits are connected in a ring form overlaps in two stages. GroEL
Captures the denatured protein in the recess of the donut structure,
Consumption of nucleotides such as TP and cofactor GroE
It has been known that the protein efficiently folds into a protein having the correct three-dimensional structure as S bonds. Some attempts have been made to apply this chaperonin to protein stabilization. For example, Japanese Patent Application Laid-Open No. 7-67641 proposes a "method of stabilizing an enzyme in a solution by adding a chaperonin protein and nucleotides such as ATP to an enzyme-containing solution". Japanese Patent Application Laid-Open No. 7-48398 discloses that "regeneration of an active protein from a chemically denatured inactive protein, an inactive protein accumulated in a transformant used for genetic manipulation, etc." For the purpose of Thermus thermophilu
s) Method using purified chaperonin ”.

[0004] Each of these uses the action of forming a three-dimensional structure of a protein possessed by a chaperonin. When the three-dimensional structure is deformed by heat or a denaturing agent, the polypeptide chain of the protein is unwound into its original three-dimensional structure (folding) ) The purpose is achieved by the action. However, chaperonins are generally ATP, CTP, UDP
It is necessary to coexist such high energy substances. It has been reported that the use of a very high concentration of chaperonin can stabilize a protein without the need for a high energy substance such as ATP, but chaperonin is expensive and lacks economy.

On the other hand, PPIase is a target molecule of cyclosporin and FK506, which are known as immunosuppressants, and has a cyclophilin type and FKBP (FK506 binding prase) based on the sensitivity to the respective immunosuppressants.
otein) type. PPIase has a function of promoting the regeneration of protein higher-order structure by catalyzing the cis-trans isomerization reaction of the peptide bond at the N-terminal side of the proline residue in the polypeptide chain. It was expected to be used for stabilization and regeneration of inactive proteins.
Unlike the chaperone, PPIase has an advantage that a high energy substance such as ATP is not required.

[0006] PPIases that have been commercially available so far are:
For example, Cyclophilin type and FKBP from Sigma
Although two types of each type are marketed, each type is derived from an animal, and its stability is low, so that it has to be stored at a low temperature of 4 ° C. or −20 ° C. In addition, all are prepared from animal organs, and there is a problem in securing a supply source. In this regard, the thermophilic eubacteria Bacillus stearothermophilus (Bacill)
Although a cyclophilin-type PPIase derived from U.S. stearothermophilus has been reported, it is inactivated at 65 ° C. and is not very thermostable. On the other hand, F
As for the heat-resistant KBP-type PPIase, the present inventors have only proposed a FKBP-type PPIase derived from methane bacteria (JP-A-10-91). The discovery of PPIase was expected.

[0007]

DISCLOSURE OF THE INVENTION In view of the above problems, an object of the present invention is to provide a thermostable FKBP-type PPIase and a gene from which it is produced.

[0008]

Means for Solving the Problems The present inventor has proposed a heat-resistant FK.
In order to obtain the entire base sequence of BP-type PPIase and the gene encoding it, P-type PPIase was prepared from the genome of a thermophilic archae belonging to the genus Aeropyrum.
A fragment of the PPIase gene was obtained by the CR method, and screening was performed from a genomic DNA library using this as a probe, a clone having the entire base sequence of the PPIase gene was obtained and its base sequence was determined. An expression product, PPIase, was obtained.
The present invention has been completed based on these findings.

That is, the present invention provides the following (a) or (b)
This is a gene that encodes a protein. (a) a protein consisting of the amino acid sequence of SEQ ID NO: 2; (b) a protein consisting of the amino acid sequence of SEQ ID NO: 2 in which one or more amino acids have been deleted, substituted or added, and a PPIase activity The present invention also relates to a protein represented by the following (a) or (b): (a) a protein consisting of the amino acid sequence of SEQ ID NO: 2; (b) a protein consisting of the amino acid sequence of SEQ ID NO: 2 in which one or more amino acids have been deleted, substituted or added, and a PPIase activity Protein with

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail. (1) Gene The gene of the present invention encodes the following protein (a) or (b).

(A) a protein comprising the amino acid sequence of SEQ ID NO: 2; (b) a protein comprising the amino acid sequence of SEQ ID NO: 2 wherein one or more amino acids are deleted, substituted or added; and Protein having PPIase activity Here, the protein (b) is obtained from the protein (a) by a technique commonly used at the time of filing the application, for example, site-directed mutagenesis (Nucleic Acids Res. 10, 6487-6500). , 19
There is no particular limitation as long as it can be produced according to (82).

The gene of the present invention can be obtained, for example, by the following procedure. First, for the purpose of obtaining the gene of the present invention, a strain belonging to the genus Aeropyram, for example, a strain belonging to Aeropyrum pernix (Aeropyrum pernix) is cultured under aerobic conditions at a temperature of about 90 ° C., and then collected, and several grams of cells are obtained. Get. For details on the culture method of this bacterium, see "Internat
ional Journal of Systematic Bacteriology Oct. (199
6) p1070-1077 ". Aeropyram pernicus strain K1 has been deposited with the RIKEN Microorganisms Preservation Line Storage Facility under the storage number JCM9820. After the obtained cells are lysed with a bacterial solubilizing agent such as SDS, genomic DNA is extracted by a technique such as phenol extraction and ethanol precipitation. This genomic DNA
After digestion with an appropriate restriction enzyme, ligated to an appropriate vector,
Create a genomic DNA library. The vector is λ
Various phage-derived vectors, for example, phagemid DNA such as λgt10 and λZAP, or plasmid vectors such as pUC18 and pBR322 can be used.

On the other hand, FKBP type P derived from another species
Based on the amino acid sequence of the region of high homology between PIase genes, the corresponding DNA is synthesized and PCR
To be used as primers. By performing PCR using the primers and the genomic DNA as a template, a partial fragment of the target gene can be obtained. The partial fragment is [
By labeling with a radioactive element such as ³² P] or a non-radioactive compound such as dicoxigenin, it can be used as a probe for gene screening.

In order to obtain the entire base sequence of the target gene, the above-mentioned genomic DNA library may be introduced into a host such as Escherichia coli, and a clone which strongly binds to the above-mentioned labeled probe may be selected. The nucleotide sequence can be determined by a general method such as the Sanger method or the Maxim-Gilbert method. According to the above procedure, the gene of the present invention including the termination codon from the translation initiation codon can be isolated. The microorganism containing the gene of the present invention has been deposited as FERMP-16987 with the Institute of Biotechnology and Industrial Technology, National Institute of Advanced Industrial Science and Technology (Deposit date: September 10, 1998).

(2) Protein The protein of the present invention is represented by the following (a) or (b). (a) a protein consisting of the amino acid sequence of SEQ ID NO: 2; (b) a protein consisting of the amino acid sequence of SEQ ID NO: 2 in which one or more amino acids have been deleted, substituted or added, and a PPIase activity Here, the protein (b) is not particularly limited as long as it can be produced from the protein (a) in the same manner as described above by a technique commonly used at the time of filing the present application. The protein of the present invention can be produced, for example, by inserting the gene of the present invention into an expression vector such as a pET system as appropriate, introducing the gene into a microorganism or cultured cells, and expressing it.

The protein of the present invention is an enzyme classified into the FKBP type in PPIase, and is an animal-derived PKBase.
Like PIase, it has the ability to bind to the immunosuppressant FK506. The action of the PPIase is an enzyme that catalyzes the cis-trans isomerization of the N-terminal peptide bond of the proline residue which is a constituent amino acid in the polypeptide chain. Since the genus Aeropyram used in the present invention can be cultured under aerobic conditions, the cultivation of the bacteria is relatively easy and the workability is excellent as compared with other anaerobic archaea. In addition, since the present bacterium exhibits an optimum growth rate under a high temperature condition of 90 to 95 ° C., PPIase obtained from these bacterium is excellent in heat resistance and is advantageous for transportation and storage.

[0017]

[Example 1]

Preparation of genomic DNA of Aeropyram pernicus Aeropyram pernicus strain K1 (hereinafter simply referred to as "K1 strain")
The conditions of Sako et al. (Int. J. Syst. Bacteriol.4
6, P.1070-1077) in a JXT medium at 90 ° C. 1 g of the obtained cells was added to 19 ml of TNE buffer (50 mM Tris-HCl pH 8.0,
The suspension was suspended in 150 mM NaCl, 100 mM EDTA) and lysed at 65 ° C. for 30 minutes in the presence of 2% SDS and 0.1 mg / ml proteinase K. Lysate is phenol / chloroform (1:
After treatment with 1) and chloroform / isoamyl alcohol (24: 1), genomic DNA was extracted by ethanol precipitation. The recovered genomic DNA was mixed with 1 ml of TE buffer (10 mM
Tris-HCl (pH 8.0, 1 mM EDTA). DNA solution
After treatment with 0.1 mg / ml RNase A at 37 ° C for 3.5 hours, 0.5%
The cells were treated with 0.1 mg / ml proteinase K for 50 minutes in the presence of SDS for 90 minutes. After adjusting the NaCl concentration to 0.7 M, 0.12 volume of 10% CTA
B (hexadecyltrimethylammonium bromide) was added, and 65
After treating at 20 ° C. for 20 minutes, the mixture was extracted with chloroform / isoamyl alcohol (24: 1), and further extracted four times with phenol / chloroform (1: 1). The DNA was precipitated with ethanol, recovered, and dissolved in 1 ml of TE buffer.

[Example 2] Amplification of the PPIase partial gene of Aeropyram pernicus by PCR Using the genomic DNA of the K1 strain prepared in Example 1 as a template and PCR using the following mixed primers,
An attempt was made to amplify the PIase gene. The DNA polymerase used was TaKaRa EX Taq polymerase (Takara). 1) FKH-F 5'-AC (ATCG) (AT) (GC) (ATCG) AT (ACT) GA (AG) GA (AG) GT-3 '2) FHK-R 5'-A (AG) ( TC) TC (GA) TG (GA) TT (GA) AA (GA) TC-3 '

A PCR fragment using the above primer set was used to obtain a DNA fragment of about 310 bp.
(Novagen) by TA cloning and dye terminatio
s by n cycle sequencing kit (Perkin Elmer)
Perform an equencing reaction, and use DNAsequencer (Applied biosyste
m company). The DNA obtained as a result of the sequence analysis was presumed to encode a part of PPIase.

Example 3 Cloning of PPIase Partial Gene of Aeropyram pernicus The genomic DNA of the K1 strain prepared in Example 1 was completely digested with BamHI, and the Sau3AI cassette (Takara) was inserted into a DNA library.
The ligation was performed by a DNA Ligase reaction using gation kit ver. 1 (Takara). Long PCR using the following primers with a part of the reaction solution as a template was performed using Takara LA Taq (Takara).

1) APCS1: 5'-TTCTTATACTCTCCGAAAGCCTCTTCAGGGGGG-3 '2) APCS2: 5'-ACATCAAGCTCCTTGAGCACCTTCTCGAGGGG-3' 3) APCS3: 5'-AGGCGAGAGGCGAGAGGTTGAAGTCCCCCC-3 '4) APCS4: 5'-AGGTGATCGAGGAGGAGG Primer C1 (Takara) 6) Cassette Primer C2 (Takara)

After PCR using a combination of APCS1 and Casset Promer C1, a part of the reaction solution was
PCR using a combination of 2 and cassette Primer C2 was performed. After collecting the amplified DNA, pT7 blue T vector (Novage
n company), transformed into Escherichia coli JM109 strain, spread on an LB plate to which 100 μg / ml of ampicillin was added, and used as a PCR product library. On the other hand, the amplified DNA obtained by PCR using APCS3 and Cassette Primer C1 followed by PCR using APCS4 and Cassette Primer C2 was ligated to pT7 blue T vector, and
A CR product library was created. The former is the PPI of the K1 strain
This is a library containing DNA encoding the N-terminal side of ase, and the latter is a library containing DNA encoding the C-terminal side.

PCR using the following primers and PCR DIG probe synthesis kit (Boehringer Mannheim)
Thus, the P1 partial gene of the K1 strain labeled with digoxigenin was amplified from the genomic DNA of the K1 strain. 1) APFK-F: 5'-GTCCGGCATTTACGATCC-3 '2) APFK-R: 5'-CCGAACCTCTCTGTAACC-3'

The above-mentioned digoxigenin-labeled probe was used for colony hybridization by the colony hybridization method.
The K1 strain PPIase gene was screened from the two PCR product libraries. Detection of positive clones
This was performed using a DIG DNA Labeling and Detection Kit (Boehringer Mannheim). After purifying the obtained positive clone, the plasmid was recovered and the inserted sequence was analyzed. The full-length PPIase gene of the K1 strain was determined from the partial gene sequence, N-terminal DNA sequence and C-terminal DNA sequence of the K1 strain obtained in Example 2. The elucidated gene sequence and deduced amino acid sequence are shown in SEQ ID NOs: 1 and 2, respectively.

[0025]

Industrial Applicability The present invention provides a thermostable FKBP-type PPIase gene that binds to the immunosuppressant FK506. P prepared from the gene of the present invention
PIases are extremely useful for regeneration of denatured proteins, stabilization of protein reagents, production of recombinant proteins, and search for new immunosuppressants and bioactive substances.

[0026]

[Sequence list] SEQ ID NO: 1 Sequence length: 804 Sequence type: Nucleic acid Number of strands: Double-stranded Topology: Linear Sequence type: Genomic DNA Sequence characteristics Origin Organism name: Aeropyrum pernix Strain name: K1 strain Sequence ATG CCG TTG AAG GAT GGA GAC TTC GTG CTG ATC AAC TAT ACT GTA AAG 48 Met Pro Leu Lys Asp Gly Asp Phe Val Leu Ile Asn Tyr Thr Val Lys 1 5 10 15 GTT GTT GAA GAC GGT TCT GAG AGG GTT ATA GAC ACG ACT CTG GAG GAA 96 Val Val Glu Asp Gly Ser Glu Arg Val Ile Asp Thr Thr Leu Glu Glu 20 25 30 GTG GCG AAG AAG TCC GGC ATT TAC GAT CCG AAG AGG GTG TAC AAG GAG 144 Val Ala Lys Lys Ser Gly Ile Tyr Asp Pro Lys Arg Val Tyr Lys Glu 35 40 45 TTC CCG GTT ATA GTG GGG AAG ACA AGC CTC CTA GGA CCC CTC GAG AAG 192 Phe Pro Val Ile Val Gly Lys Thr Ser Leu Leu Gly Pro Leu Glu Lys 50 55 60 GTG CTC AAG GAG CTT GAT GTA GGC GAG AGG CGA GAG GTT GAA GTC CCC 240 Val Leu Lys Glu Leu Asp Val Gly Glu Arg Arg Glu Val Glu Val Pro 65 70 75 80 CCT GAA GAG GCT TTC GGA GAG TAT AAG AAG GAG CTG GTT GTC AGG GTG 288 Pro Glu Glu Ala Phe Gly Glu Tyr Lys Lys Glu Leu Val Val Arg Val 85 90 95 CCC GTG AAG AGG CTT AGG AGG ATG AAC ATA CCC GTG AGG GTT GGG GAG 336 Pro Val Lys Arg Leu Arg Arg Met Asn Ile Pro Val Arg Val Gly Glu 100 105 110 GAG GTT GAG GCG GGT GGC AGA AGA GGT AAG ATA ATA AGG GTT ACA GAG 384 Glu Val Glu Ala Gly Gly Arg Arg Gly Lys Ile Ile Arg Val Thr Glu 115 120 125 AGG TTC GCC TTC ATA GAC TTC AAC CAC CCC CTG GCA GGG AAG AAG CTT 432 Arg Phe Ala Phe Ile Asp Phe Asn His Pro Leu Ala Gly Lys Lys Leu 130 135 140 AAG ATA GAG GTT GAG GTT GTA GGA AAG GTG GAT AGC GTC GAG GAC AAA 480 Lys Ile Glu Val Glu Val Val Gly Lys Val Asp Ser Val Glu Asp Lys 145 150 155 160 GCC AGG ATC CTG GCG GCC AGA GCC CTA GGC TTG GAC CCC GAC AAG ATC 528 Ala Arg Ile Leu Ala Ala Arg Ala Leu Gly Leu Asp Pro Asp Lys Ile 165 170 175 CAT GCG GAG GTT AGA GAG GGG GGC AAG GAG ATA ACC CTA AAG CTG CCC 576 His Ala Glu Val Arg Glu Gly Gly Lys Glu Ile Thr Leu Lys Leu Pro 180 185 1 90 CCG GAG GTC CTC GGG CTC AGC GAC CTC GAC GCG CTC CTG GCA AAG GCG 624 Pro Glu Val Leu Gly Leu Ser Asp Leu Asp Ala Leu Leu Ala Lys Ala 195 200 205 GTA TCA GAT GTT AGA GAG TAC CTG TCC CCT AGG AAA GTT GAC GTG GTC 672 Val Ser Asp Val Arg Glu Tyr Leu Ser Pro Arg Lys Val Asp Val Val 210 215 220 ATA TCC ATC GAG TTC CCG GAG GAG GCG CGG GAG GAG GCT GAA GCA TCT 720 Ile Ser Ile Glu Phe Pro Glu Glu Ala Arg Glu Glu Ala Glu Ala Ser 225 230 235 240 GAT ACT GGT GAG GGA GGA GAA GCG GAG GGT GAG TCG GAG CAG GCT GCA 768 Asp Thr Gly Glu Gly Gly Glu Ala Glu Gly Glu Ser Glu Gln Ala Ala 245 250 255 GAG CCC GGT GGA GGG GAA GAG AGG CAG GAG GGC TAG 804 Glu Pro Gly Gly Gly Glu Glu Arg Gln Glu Gly 260 265

SEQ ID NO: 2 Sequence length: 267 Sequence type: amino acid Topology: unknown Sequence type: protein Sequence characteristics Origin Organism name: Aeropyrum pernix Strain name: K1 strain Sequence Met Pro Leu Lys Asp Gly Asp Phe Val
Leu Ile Asn Tyr Thr Val Lys 15
10 15 Val Val Glu Asp Gly Ser Glu Arg Val
Ile Asp Thr Thr Thr Leu Glu Glu 20 25
30 Val Ala Lys Lys Ser Gly Ile Tyr Asp
Pro Lys Arg Val Tyr Lys Glu 35 40
45 Phe Pro Val Ile Val Gly Lys Thr Ser
Leu Leu Gly Pro Leu Glu Lys 50 55
60 Val Leu Lys Glu Leu Asp Val Gly Glu
Arg Arg Glu Val Glu Val Pro 65 70
75 80 Pro Glu Glu Ala Phe Gly Glu Tyr Lys
Lys Glu Leu Val Val Arg Val 85
90 95 Pro Val Lys Arg Leu Arg Arg Met Asn
Ile Pro Val Arg Val Gly Glu 100 105
110 Glu Val Glu Ala Gly Gly Arg Arg Gly
Lys Ile Ile Arg Val Thr Glu 115 120
125 Arg Phe Ala Phe Ile Asp Phe Asn His
Pro Leu Ala Gly Lys Lys Leu 130 135
140 Lys Ile Glu Val Glu Val Val Gly Lys
Val Asp Ser Val Glu Asp Lys 145 150
155 160 Ala Arg Ile Leu Ala Ala Arg Ala Leu
Gly Leu Asp Pro Asp Lys Ile 165
170 175 His Ala Glu Val Arg Glu Gly Gly Lys
Glu Ile Thr Leu Lys Leu Pro 180 185
190 Pro Glu Val Leu Gly Leu Ser Asp Leu
Asp Ala Leu Leu Ala Lys Ala 195 200
205 Val Ser Asp Val Arg Glu Tyr Leu Ser
Pro Arg Lys Val Asp Val Val 210 215
220 Ile Ser Ile Glu Phe Pro Glu Glu Ala
Arg Glu Glu Ala Glu Ala Ser 225 230
235 240 Asp Thr Gly Glu Gly Gly Glu Ala Glu
Gly Glu Ser Glu Gln Ala Ala 245
250 255 Glu Pro Gly Gly Gly Glu Glu Arg Gln
Glu Gly 260 265

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat (Reference) C12R 1:01) (72) Inventor Tadashi Maruyama 75-1, Hirata 3rd Land 75-1, Kamaishi City, Iwate Pref. (72) Inventor Masahiro Furuya 2-1 Momoyama, Shimamoto-cho, Mishima-gun, Osaka Prefecture F-term (reference) in Sekisui Chemical Co., Ltd. 4B024 AA01 AA20 BA07 CA03 HA01 4B050 CC01 CC04 LL01 LL10

Claims (2)

[Claims] 1. A gene encoding the following protein (a) or (b): (a) a protein consisting of the amino acid sequence of SEQ ID NO: 2; (b) a protein consisting of the amino acid sequence of SEQ ID NO: 2 in which one or more amino acids have been deleted, substituted or added, and peptidylprolyl Protein having cis trans isomerase activity 2. A protein represented by the following (a) or (b): (a) a protein consisting of the amino acid sequence of SEQ ID NO: 2; (b) a protein consisting of the amino acid sequence of SEQ ID NO: 2 in which one or more amino acids have been deleted, substituted or added, and peptidylprolyl Protein having cis trans isomerase activity