JPH09313184A - Cyclophilin-type PPIase gene derived from halophilic archaea - Google Patents

Abstract

(57) Abstract: Regeneration of denatured protein, stabilization of protein reagent, production of recombinant protein, and further novel immunosuppressant,
A gene encoding PPIase, which is an enzyme useful for the development of physiologically active substances. [Effect] Similar to those derived from animals, a novel cyclophilin-type PPIase gene, which is capable of binding to the immunosuppressant cyclosporin A, is provided.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an enzyme useful for regeneration of denatured proteins, stabilization of protein reagents, improvement of recovery rate in the production of recombinant proteins, development of novel immunosuppressive agents, and physiologically active substances. PPIase (Peptidyl pr
olyl cis-trans isomerase). The protein obtained by culturing a microorganism or a cultured cell containing a recombinant DNA incorporating the present gene in a culture medium and collecting the protein accumulated in the culture can be used for various purposes. You can

[0002]

BACKGROUND OF THE INVENTION Proteins are composed of polypeptides,
In recent years, attention has been paid to factors that act on the formation and maintenance of the structure.
Molecular chaperones, PPIases, etc. have come to be known as the factors.

In the production of useful proteins by gene recombination technology, the target protein may be produced as an inactive inclusion body due to overproduction in an expression system such as Escherichia coli [for example, basic course of microbiology 8 genetic engineering, pp73-74. ,
Tadahiko Ando / Kenji Sakaguchi, Kyoritsu Shuppan, 1987]. In order to activate such an inactivated protein, a method of recovering an inactive inclusion body, solubilizing it with a denaturing agent, coexisting these factors during a regeneration reaction by dilution, and a gene encoding these factors In addition, a method for expressing a target gene has been considered. On the other hand, it is desired that the protein reagent be liquefied and stored at room temperature, and it is considered possible to achieve it by coexisting these factors.

On the other hand, PPIase is a target molecule of immunosuppressant cyclosporin A and FK506, and is involved in intracellular protein folding and intracellular signal transduction system. It is thought that the search for the drug will lead to the development of new immunosuppressants and other physiologically active substances.

The commercially available PPIases are:
For example, Cyclophilin from Sigma
Cyclosporin A binding protein) type and FKB
Two types of P (FK506-binding protein) type are on the market, one type each (Sigma 1996)
Both the annual catalogs P.311 and P.447) were prepared from animal organs, and there was a problem in securing a supply source. E. coli and Bacillus stearotherm, which is a thermophilic eubacterium
A cyclophilin-type PPIase from ophilus has been reported [Journal of Fermentation and Bioeng
ineering, Vol.79, No.2, 87-94, 1995] are not sensitive to cyclosporin A, and thus are different from the animal type, and are considered to be difficult to use for immunosuppressant drug search. From this, the discovery of a novel PPIase sensitive to cyclosporin A was expected.
The present inventors have found that Halobacterium cutirub belonging to archaea
A cyclophilin-type PPIase was purified from rum and reported to be sensitive to cyclosporin A [Biochemical and Biophysical Research Communicat
ion, Vol.198, No.2, 87-94, 1994]. However,
There was a problem in obtaining PPIase, for example, 4 days were required for culturing the bacterium, and 5 purification steps were required after crushing the cells.

[0006]

SUMMARY OF THE INVENTION It is an object of the present invention to provide a gene which is a source of production of cyclophilin type PPIase so as to produce it by using a genetic engineering technique.

[0007]

[Means for Solving the Problems] Cyclosporin A-sensitive cyclophilin-type PPIase (hereinafter simply referred to as "P
In order to obtain the entire sequence of the gene encoding "PIase"), the purified protein is isolated, the N-terminal amino acid sequence is determined, a primer is synthesized based on these sequences, and PCR is performed.
The present invention was completed by obtaining a fragment of this gene by the method and screening a genomic DNA library using this as a probe to obtain a clone encoding the full-length PPIase gene and determining its nucleotide sequence.

That is, the present invention provides D represented by SEQ ID NO: 1.
It is a PPIase gene having a DNA base sequence substantially identical to the NA base sequence or the DNA base sequence represented by SEQ ID NO: 1.

Hereinafter, the present invention will be described in detail. The PPIase gene of the present invention has a DNA base sequence represented by SEQ ID NO: 1 or a DNA base sequence substantially the same as the DNA base sequence represented by SEQ ID NO: 1. Here, "DN substantially identical to the DNA base sequence represented by SEQ ID NO: 1"
"A nucleotide sequence" refers to deletion, substitution, deletion or substitution of some nucleotide residues of the DNA nucleotide sequence represented by SEQ ID NO: 1.
It is a nucleotide sequence that encodes a polypeptide that can act as a PPIase, which is a sequence that has undergone changes such as additions.

The gene of the present invention can be obtained by the following procedure. First, microorganisms capable of producing PPIase, such as Halobacterium
cutirubrum) After culturing the DSM669 strain and the like, collecting the cells, crushing the cells, and then purifying the PPIase by various chromatography. The purified enzyme is separated by SDS polyacrylamide gel electrophoresis and electrically transferred to a hydrophobic PVDF membrane or the like. Coomassie Brilliant Blue R-25
After cutting out a band detected with a dye such as 0, the amino acid sequence from the N terminus is determined by a protein sequencer. A partial gene of PPIase can be obtained by designing a primer used for PCR from the N-terminal amino acid sequence and performing PCR using genomic DNA derived from a microorganism capable of producing PPIase as a template. This is labeled with a non-radioactive probe such as [ ³² P] or dicoxygenin,
Used as a probe for gene screening.

On the other hand, the genomic DNA of a microorganism capable of producing PPIase is cleaved with an appropriate restriction enzyme and then ligated to an appropriate vector to prepare a genomic DNA library. As the vector, various vectors derived from λ phage, for example, λgt
A plasmid vector such as 10 or Zapll, or pUC18 or pBR322 can be used. To select a clone carrying the target gene, hybridization may be carried out using the above-mentioned probe, and a clone that strongly binds to this may be selected. In addition, PCR is performed by appropriately synthesizing a primer from the amino acid sequence of a region having high homology between cyclophilin-type PPIase genes derived from other organisms.
A DNA fragment may be obtained by the method and used as a probe. The nucleotide sequence can be determined by a general method such as Sanger method or Maxam-Gilbert method. By the above procedure, the full-length DNA encoding PPIase including the translation initiation codon to the termination codon can be isolated. Escherichia coli pTPE1-15 containing this PPIase-encoding DNA has been deposited as FERM P-15621 at the Institute of Biotechnology, Institute of Industrial Science and Technology (deposit date May 15, 1996). The isolated DNA can be inserted into an appropriate expression vector, introduced into a microorganism or cultured cells, and expressed, whereby a large amount of the protein can be prepared.

[0012]

BEST MODE FOR CARRYING OUT THE INVENTION

[0013]

The present invention will be described below with reference to examples, but the scope of the present invention is not limited to these examples. [Example 1] Analysis of N-terminal amino acid sequence of PPIase derived from strain DSM 669 [Reference to Nagashima et al. [Biochemical and Biophysical Research]
Communication, Vol.198, No.2, 87-94, 1994], according to the method described in Halobacterium kutilbulum DSM669.
The strain was cultured and PPIase was purified. This enzyme is 14% SD
After S polyacrylamide gel electrophoresis, it was electrically blotted on a PVDF membrane (Applied Biosystems) (Japan Edo Co., Ltd.) and stained with CBB-R250. The cut out 31kDa band is the protein sequencer PS
33 residues of the N-terminal amino acid sequence were determined by Q-2 (Shimadzu Corporation). The sequence is shown below. Pro-Val-Asp-Ala-Thr-Val-His-Thr-Ser-Glu-Gly-Glu-Ph
e-Asp-Ile-Glu-Leu-Tyr-Asp-Glu-Arg-Ala-Pro-Xaa-Thr-
Val-Glu-Asn-Phe-Leu-Xaa-Leu-Ala (Xaa: unidentified amino acid)

Example 2 Preparation of Genomic DNA of DSM669 Strain Document [Biochemcal and Biophysical Research Communic
ation, Vol.198, No.2, 87-94, 1994].
75% casamino acid, 1% yeast extract, 0.3% citric acid,
0.2% potassium chloride, 2% magnesium sulfate, 0.005%
The DSM669 strain was cultivated using 200 ml of ferrous sulfate, 0.00002% manganese sulfate, 25% sodium chloride, pH 7.4).

0.6 g of wet cells were washed twice with 10 ml of 3.9 M NaCl solution, suspended in 10 ml of 1 mM Tris-HCl buffer containing 0.1 mM EDTA, and shaken at room temperature for 4 hours to lyse the cells. Then, the temperature was raised to 50 ° C and proteinase K solution (20
(50 mg / ml) was added and shaken for 4 hours. After phenol treatment and chloroform treatment (solution amount 10 ml), 1 ml of 3M sodium acetate solution (pH 5.2), 25 ml of ethanol were added, and the mixture was left at -20 ° C for 2 hours. Then, centrifuge with a high speed centrifuge to precipitate the DNA, wash the precipitate with 3 ml of 70% ethanol solution, dry to dryness with a centrifugal evaporator, and add TE buffer solution.
Dissolved in 1.5 ml. 50μ of RNase A solution (0.5mg / ml)
1 was added and left at 37 ° C. for 2 hours. Add 10% SDS solution to 0.
15 ml and 4 μl of proteinase K solution (20 mg / ml) were added and left at 50 ° C. for 2 hours. After phenol / chloroform treatment and ethanol precipitation were performed again, the precipitate was washed with 70% ethanol solution and dried by centrifugal evaporator to obtain T
It was dissolved in 1 ml of E buffer. By this operation, about 0.1 mg of genomic DNA was obtained.

[Example 3] Amplification of PPIase gene fragment by PCR and determination of its nucleotide sequence The following DNA primers were synthesized from the N-terminal amino acid sequence of PPIase obtained in Example 1. Hppi-1s is the position corresponding to the amino acid sequence His-Thr-Ser-Glu-Gly-Glu-Phe-Asp, and Hppi-1a is the amino acid sequence Glu-Asn-Phe-Leu-Xa.
The position corresponding to a-Leu-Ala was designed as the opposite strand. Hp
An unidentified amino acid (Xaa) in the sequence of pi-1a was designed as Ala. Hppi-1s: CA (TC) AC (ATCG) (AT) (CG) (ATCG) GA (AG) GG (ATC
G) GA (AG) TT (TC) GA Hppi-1a: GC (ATCG) A (AG) (ATCG) GC (ATCG) A (AG) (AG) AA (A
G) TT (TC) TC

A PCR reaction was carried out using the PCR Amplification kit (Takara Shuzo) with the genomic DNA of the DSM 669 strain prepared according to Example 2 as a template. There is a Tm value of the primer as a condition for determining the annealing temperature, and it is said that it is preferable to set the temperature lower by about 5 ° C. (for example, the latest technology of PCR method, Takeshi Hayashi, Ed. 1995), but both are mixed primers, 76 ℃ ~ 60 ℃ in 1s, 66 ℃ ~ 50 ℃ in 1a. Therefore, the annealing temperature was 53 ° C and 50 ° C.
No amplification of NA fragment was observed. Then, at 45 ° C., a fragment of the expected length (80 bp) was amplified. On the other hand, at 37 ° C, the number of amplified fragments increased to 4 to 5. Therefore 45
It was decided to analyze the sequence using the fragment amplified at ° C.

A method using the above 80 bp DNA fragment with low melting point agarose (Lab Manual, Genetic Engineering Supplement, p28
(Masami Muramatsu, Maruzen, 1990), pCR-Scri
Using the ptSK (+) Cloning Kit, ligate to the pCR-ScriptSK (+) vector (Stratagene), and competent cell E.co.
li JM109 was used for transformation. After selecting a positive clone and confirming that the insert fragment is present, 25 ml of LB medium (50
(containing μg / ml of ampicillin) and cultured overnight at 37 ° C. with shaking. After the culture solution was centrifuged to collect the cells, plasmid DNA was prepared mainly according to the method described in Lab Manual Genetic Engineering Supplement, pp.51-52 (edited by Masami Muramatsu, Maruzen, 1990). By this operation, about 20 μg of DNA was prepared.
Dye was used with 0.5 μg of DNA as a template per sample.
The terminator cycle sequencing kit (Perkin Elmer) was used to perform a sequencing reaction with the primers on the vector side, and the nucleotide sequence was determined with a DNA sequencer (373A, Applied Biosystems). As a result, the gene fragment amplified by PCR had the following sequence. CACACAACGGAAGGGGAGTTCGA CATCGAACTGTACGACGAGCGGGCCCCCCGCACCGTC GAGAACTTCC HTTEGEFDIELYDERAPRTVE NFL TCGCCCTCGC AL

The underlined portion is the amplified portion of the primer, and there is a high possibility that it does not completely match the original sequence.
As compared with the results of Example 1, S has a sequence encoding T at the 3rd amino acid of the 5′-side primer, and the unidentified amino acid portion designed as A of the 3′-side primer has a sequence encoding A. Met. The internal unidentified amino acid was R. All other sequences were in agreement with the results of Example 1 and were judged to be a part of this PPIase gene.

Example 4 Preparation of DSM669 Strain Genomic DNA Library To 4 μg of the genomic DNA prepared according to Example 2, 1.5 units of restriction enzyme Sau3AI was added, and the mixture was reacted at 37 ° C. for 1 hour for cleavage. The solution after the cleavage reaction was subjected to a precipitation treatment with phenol and ethanol to obtain DNA. On the other hand, BamHI
Cleaved and dephosphorylated pUC18 was purchased from Pharmacia and a genomic DNA fragment was inserted at the digestion position Takara.
Ligation kit Ver. 1 was ligated and transformed with E. coli JM109 to obtain a Sau3AI genomic DNA library.

Also, 18 units of the restriction enzyme BamHI was added to 4 μg of genomic DNA, and the mixture was reacted at 37 ° C. for 18 hours for cleavage. The solution after the cleavage reaction was subjected to a precipitation treatment with phenol and ethanol to obtain DNA. Then, ligated to pUC18 that had been digested with BamHI and dephosphorylated in the same manner as above,
A BamHI genomic DNA library was created.

Example 5 Selection of Recombinant Clone Containing PPIase Gene As a probe for obtaining the full-length PPIase gene, first, a sequence of 60 bp on the pCR-ScriptSK (+) vector side was included.
DIG DNA labeling and detection of 140bp DNA fragment
The kit was designed by using a kit (Boehringer Mannheim) with random primers, and primers based on the 5'side and 3'side sequences of the 80 bp DNA fragment prepared in Example 3 were designed, and DIG DNA labeling an
A probe labeled with a random primer using a d detection kit (Boehringer Mannheim) was used. However, in both cases, the labeling efficiency was low, and particularly in the former case, a good probe could not be prepared because it contained the sequence of Betacu. Therefore, PCR DIG probe synthesis
PCR using a kit (Boehringer Mannheim)
A DIG-labeled probe was prepared.

On the other hand, the transformant prepared with Sau3AI according to Example 4 was plated on an LB plate (including ampicillin and X-gal), and after overnight culture, it was fixed on a nylon membrane High Bond N (Amersham) to give a colony. Hybridization was performed. The method is mainly DIG DNA labellin
The protocol attached to the g and detection kit (Boehringer Mannheim) was followed. Various studies were conducted on the conditions for pre-hybridization, hybridization and filter washing. When the temperature was 68 ° C, the composition of the washing buffer solution was 0.5 x SSC (containing 0.1% SDS) or
No positive clone was found in 0.1 × SSC (containing 0.1% SDS). Next, prehybridization,
Hybridization at 62 ℃, washing at 60 ℃ 0.1 x S
When using SC (containing 0.1% SDS), about 8,
Two positive clones were obtained from 000 clones. However, neither of them contained the full length sequence encoding PPIase, and was cleaved at the Sau3AI site existing inside the gene.

Next, screening was carried out using the library prepared with BamHI according to Example 4, and 3 colonies binding to the probe were obtained from about 5000 clones. D inserted in the plasmid of these colonies
As a result of examining the NA fragment, it was revealed that two clones contained the full-length PPIase gene. And Dye Te
A sequencing reaction was performed using the rminator cycle sequencing kit, and the nucleotide sequence was determined with a 373A DNA sequencer. In this case, since the GC content was as high as 69% and there were many continuous G or C sequences, the primer was selected to be a sequence that is not biased to GC and does not have a secondary structure. The following three types of primers were used on the Forward side. The name of the primer, the position of the sequence (the number when the start codon A is number 1), and the nucleotide sequence are listed in this order.

Hppi-3F: 47 to 65: TCGAACTGTACGACGAGCG Hppi-4F: 326 to 344: CCGGCGTGCTCTCGATGGC Hppi-5F: -321 to -303: CCGAGTACGGCATGGACTT (5'upstream side) The following 3 types of Reverce side primers were used. . Hppi-3R: 100 to 82: CGAGGTTCAGGAAGTTCTC Hppi-4R: 497 to 479: CGTCGATGTTGTGCTGGAG Hppi-5R: 712 to 684: GCGTCGTTGGCGTCGGTCT (3'downstream) All the sequences were determined using these primers. The gene obtained and the amino acid sequence deduced therefrom are shown in FIG.
Shown in The position of the methionine residue, which is the translation initiation codon, was estimated from the amino acid sequence determined in Example 1.

[0026]

INDUSTRIAL APPLICABILITY The present invention provides a novel cyclophilin-type PPIase gene, which has a binding property to the immunosuppressant cyclosporin A as well as those derived from animals.
The protein produced from the gene of the present invention is extremely useful for regeneration of denatured protein, stabilization of protein reagents, production of recombinant protein, and search for new immunosuppressants and bioactive substances.

[0027]

[Sequence list]

SEQ ID NO: 1 Sequence length: 543 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: Genomic DNA Origin organism name: Halobacterium cutiru
brum

[Brief description of drawings]

FIG. 1 is a diagram showing a DNA base sequence of the PPIase gene and an amino acid sequence encoding the DNA base sequence.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Tokuro Iwabuchi 75-1 Hirata No. 3 75-1 Hirata, Kamaishi-shi, Iwate Stock Company, Kamaishi Research Institute, Marine Biotechnology Research Institute (72) Masaru Maruyama Sodeshi-cho, Shimizu-shi, Shizuoka Prefecture 1900 Kaiyo Biotechnology Research Institute Co., Ltd. Shimizu Research Institute

Claims (1)

[Claims] 1. A DNA base sequence represented by SEQ ID NO: 1,
Alternatively, a PPIase gene having a DNA base sequence substantially identical to the DNA base sequence represented by SEQ ID NO: 1.