JPH1091A - FKBP-type PPIase derived from thermophilic archaea and gene encoding the same - Google Patents

Abstract

(57) [Summary] P is a highly heat-resistant enzyme which is useful for regeneration of denatured proteins, stabilization of protein reagents, production of recombinant proteins, and development of novel immunosuppressants and bioactive substances.
PIase and the gene encoding it. The present invention provides a PPIase useful for regenerating a denatured protein, stabilizing a protein reagent, producing a recombinant protein, searching for a novel immunosuppressant, a bioactive substance, and the like, and a gene encoding the same.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to an enzyme having high heat resistance and useful for regeneration of a denatured protein, stabilization of a protein reagent, production of a recombinant protein, and development of a novel immunosuppressant and a physiologically active substance. PPIase (Peptidyl prolyl cis
-trans isomerase) and the gene encoding it. A protein obtained by culturing a microorganism or a cultured cell containing a recombinant DNA incorporating the present gene in a culture solution and collecting the protein accumulated in the culture,
It can be used for various applications.

[0002]

2. Description of the Related Art Proteins are polypeptides, and factors that act on the formation and maintenance of their structures have attracted attention in recent years. That is the molecular chaperone, PPIase. In the production of useful proteins by genetic recombination techniques, in a system such as Escherichia coli, the target protein may be produced as an inactive inclusion body due to overproduction. 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, protein reagents are desired to be liquefied and stored at room temperature, but it is thought that they can be achieved by coexisting these factors.

[0003] On the other hand, PPIase is a target molecule of the immunosuppressive agents cyclosporin A and FK506, and is involved in protein folding and intracellular signal transduction in cells. It is thought that this will lead to the development of new immunosuppressants and other bioactive substances.

[0004] Commercially available PPIases include:
For example, from Sigma, Cyclophilin (Cyclosporin A)
Binding protein) type and FKBP (FK506 binding protein) type are now on the market, one by one (Sigma Catalog 1996, p. 311 and
P.447) are all derived from animals and their stability is low, and they must 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, [Jo
urnal of Fermentation and Bioengineering, Vol. 79,
No. 2, 87-94, 1995], Bacillus, a thermophilic eubacterium
Cyclophilin type PPI from stearothermophilus
Although ase has been reported, inactivation occurs at 65 ° C., and the heat stability is not so high. In addition, since it is not sensitive to cyclosporin A, it differs from the animal type, and is considered to be difficult to use for searching for immunosuppressants. On the other hand, thermostable FKBP-type PPIase has not been found so far. From this, the discovery of a new PPIase having higher thermostability was expected.

[0005]

SUMMARY OF THE INVENTION An object of the present invention is to provide a gene that is a source of production of thermostable FKBP-type PPIase by using genetic engineering techniques.

[0006]

In order to obtain the entire sequence of the gene encoding the PPIase of the present invention, a purified PPIase is isolated, its N-terminal amino acid sequence, and the amino acid sequence of the peptide produced by protease digestion. Is determined, primers are synthesized based on those sequences, and PC
A fragment of the PPIase gene is obtained by the R method, a genomic DNA library is screened using the fragment as a probe, a clone having the entire base sequence of the PPIase gene is obtained, and its base sequence is determined.
The present invention has been completed.

That is, the present invention relates to the D
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. The present invention also relates to a PPIase having the amino acid sequence represented by SEQ ID NO: 2 or an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 2.

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 identical to 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.
A sequence in which a change such as addition has occurred, and refers to a nucleotide sequence encoding a polypeptide that can act as a PPIase.

[0009] The PPIase of the present invention has the amino acid sequence represented by SEQ ID NO: 2 or an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 2.
Here, “an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 2” refers to deletion, substitution, addition, etc. of some amino acid residues of the amino acid sequence represented by SEQ ID NO: 2. Is an amino acid sequence representing a polypeptide that can act as a PPIase.

[0010] The PPIase of the present invention is classified into the FKBP type, and has an ability to bind to FK506 similarly to animal-derived PPIase. In addition, it has heat resistance and is hardly deactivated even at high temperatures. Such a PPIase has not been known so far,
Aase is a novel enzyme.

The gene of the present invention can be obtained by the following procedure. First, a microorganism capable of producing thermostable PPIase,
For example, after culturing Methanococcus thermolithotrophicus DSM2095 strain, etc., collecting cells, crushing the cells, and purifying PPIase by various chromatography. The purified PPIase is separated by SDS polyacrylamide gel electrophoresis, and is electrically transferred to a hydrophobic membrane such as a PVDF membrane. After a band detected by a dye such as Coomassie brilliant blue R-250 is cut out, the amino acid sequence from the N-terminus is determined using a protein sequencer. The excised band is digested with a suitable protease such as lysyl endopeptidase, and the amino acid sequence of the peptide recovered by reverse phase chromatography is determined in the same manner. Primers used for PCR are designed from the N-terminal amino acid sequence and the amino acid sequence of the recovered peptide, and PCR is performed using a genomic DNA derived from a microorganism capable of producing a thermostable PPIase as a template to obtain a partial gene of PPIase. . This is labeled with a non-radioactive probe such as [ ³² P] or dicoxygenin and used as a probe for gene screening.

On the other hand, genomic DNA of a microorganism capable of producing a thermostable PPIase is digested 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, such as λgt10 and λZapII, or plasmid vectors such as pUC18 and pBR322 can be used. To select a clone holding the target gene, hybridization may be performed using the probe, and a clone that strongly binds to the probe may be selected. Also, FK derived from other organisms
PCR is performed by appropriately synthesizing primers from the amino acid sequence of a region having a high homology between BP type PPIase genes.
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 the Sanger method or the 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 pUFKC1 containing this PPIase-encoding DNA was submitted to
Deposited as FERM P-15622 (Deposit date: May 1996
March 15). 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.

[0013]

BEST MODE FOR CARRYING OUT THE INVENTION

[0014]

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 Isolation and Purification of PPIase from DSM2095 Strain A thermophilic archaeon, Methanococcus thermolysortrophys strain DSM2095 (hereinafter, simply referred to as "DSM2095 strain") was transformed into a seawater-based medium (yeast extract). 2g, bactotripton 2
g, sodium acetate 1 g, PIPES 1.7 g, DAB
Vitamin solution (Appl.Environ.Microbial., 55, 2832-
2836, 1989] 65 ° C. under anaerobic conditions and aeration of a mixed gas of hydrogen / carbon dioxide (4: 1) in 10 ml / L seawater pH 6.8)
And the cells were collected overnight. By suspending this in a 25 mM phosphate buffer (pH 7.0), the cells are disrupted by osmotic shock. The supernatant is collected by centrifugation, and this is collected with a Butyl scpharose column (Pharmacia)
And subjected to hydrophobic chromatography using the above method to collect a PPIase-active fraction. PPIase activity [Nature, Vol.
337, No. 6206, 473-475, 1989] can be measured by the chymotrypsin coupling method. The recovered active fraction is passed through an ultrafiltration membrane (fraction molecular weight 10
000) and separated by gel filtration using a Superose 12HR column (Pharmacia). The fractions thus collected were then subjected to anion exchange chromatography on a MonoQ column (Pharmacia). The recovered active fraction is finally TSKgel Ethr-5P
Separation was performed by hydrophobic chromatography using a W column (Tosoh). For the collected fraction, SD
S polyacrylamide gel electrophoresis was performed. The results are shown in FIG. The right side is the collected fraction, and the left side is the molecular weight marker. As shown in FIG. 1, only the band of the PPIase having a molecular weight of about 16 kD can be clearly detected on the gel. Therefore, it is considered that impurities were almost completely removed in this purification step.

The FPI of the PPIase purified as described above
Binding to K506 was determined from its inhibition of PPIase activity. The results are shown in FIG. As shown in FIG.
PPIase is similar to FKBP derived from animals as well as FK506.
It was binding. The IC ₅₀ value was 250 nM.

Further, the thermostability of PPIase was examined using its enzyme activity as an index. The results are shown in FIG. In the figure, the circle indicates the case where the heating was performed at 100 ° C., and the circle indicates the case where the heating was performed at 90 ° C. As shown in FIG. 3, the half-life of the activity is 30 minutes.
(100 ° C.) and 90 minutes (90 ° C.), indicating that the protein is extremely thermostable.

Example 2 Analysis of Partial Amino Acid Sequence of PPIase The PPIase purified in Example 1 was further separated by 20% SDS polyacrylamide gel electrophoresis. Then, it was electrically blotted on a PVDF membrane (Applied Biosystems) (Nippon Edo), stained with Coomassie Brilliant Blue R-250, and dyed at 16 kDa.
Protein was detected. The N-terminal amino acid sequence of the excised 16 kDa band was determined using a protein sequencer PSQ-2 (Shimadzu Corporation). Also, the excised band was digested with lysyl endopeptidase (Wako Pure Chemical Industries, Ltd.) in a 25 mM Tris-hydrochloric acid (pH 8.0) solution containing 8% acetonitrile for 24 hours at 37 ° C., and the peptide recovered by sonication was micronized. Bondasphere
Separation was performed by reversed-phase chromatography using a C18 column (Waters), and the obtained three peptides were subjected to analysis using the above protein sequencer. The determined sequences are as follows.

[N-terminal amino acid sequence] Val-Asp-Lys-Gly- Lys-Ile-Lys-Val-Asp-Tyr-Ile-Gly- Ly
s-Leu-Glu-Ser-Gly-Asp-Val-Phe-Asp-Thr-Ser-Ile-Glu-
Glu (amino acid sequence of peptide purified by lysyl endopeptidase digestion) Lys- Ile-Pro-Arg-Asp-Ala-Phe-Lys Lys-Ala-Tyr-Gly-Asn-Arg-Asn- Glu-Met-Leu- Ile-Gln-Ly
s Lys-Asp-Leu-Val-Phe-Thr-Ile-Lys In addition, the underlined part indicates the design site of the PCR primer.

Example 3 Preparation of Genomic DNA of DSM2095 Strain The DSM2095 strain was cultured in a 5 L seawater-based medium in the same manner as in Example 1. 1.0 g of the wet cells was added to 10 ml of TNE buffer solution (20 mM containing 100 mM NaCl and 20 mM EDTA).
mM Tris-HCl buffer, pH 8.0). 10% S
1 ml each of the DS solution and the 1% Triton X-100 solution was added, and the mixture was left at 4 ° C. overnight. Then, the temperature was raised to 50 ° C., 50 μl of proteinase K solution (20 mg / ml) was added, and the mixture was shaken for 4 hours. After phenol treatment and chloroform treatment, 50 μl of RNase A solution (0.5 mg / ml) was added, and the mixture was allowed to stand at 37 ° C. for 1 hour. Again, 1 ml of a 10% SDS solution and 50 μl of a proteinase K solution (20 mg / ml) were added and left at 50 ° C. for 70 minutes. After phenol treatment and chloroform treatment (solution volume: 10 ml), 1 ml of a 3M sodium acetate solution (pH 5.2) and 25 ml of ethanol were added, and the mixture was allowed to stand at -20 ° C for 2 hours. Thereafter, the DNA was precipitated by centrifugation with a high-speed centrifuge, washed with 3 ml of a 70% ethanol solution, dried by a centrifugal evaporator, and dissolved in 0.5 ml of TE buffer. By this operation, about 2 mg of genomic DNA was obtained.

Example 4 Amplification of PPIase Gene Fragment by PCR and Determination of Its Base Sequence The N-terminal amino acid sequence of the PPIase obtained in Example 1 and the amino acid sequence corresponding to the amino acid sequence of the lysyl endopeptidase digested peptide were as follows: Was synthesized. PN-F1: AA (AG) AT (ATC) AA (AG) GT (ATCG) GA (TC) TA (TC) AT PF-R1: TT (TC) TG (ATG) AT (ATCG) A (AG) CAT (TC) TC (AG) TT PF-R2: TT (AG) AA (ATCG) GC (AG) TC (TC) CT (ATCG) GG (ATG)
AT

PN-F1 is a primer designed based on the N-terminal amino acid sequence, and PF-R1 and PF-R2 are primers designed based on the amino acid sequence of the lysyl endopeptidase digested peptide. DS prepared as in Example 2
PCR Amplification kit using DNA of M2095 strain as template
PCR results using (Takara Shuzo) showed that PN-F1 and PF-R1
240 bp in the combination of the primers, PN-F1 and PF-R
In the combination of 2, a 270 bp fragment was amplified. Each was electrophoresed on a 2.5% agarose gel (New Sieve GTG, Takara Shuzo), a DNA fragment was cut out, inserted into a dialysis tube, eluted by electrophoresis, and DNA was recovered by phenol treatment and ethanol precipitation. The precipitate was washed with 70% ethanol, dried under reduced pressure, and dissolved in a TE buffer.

The DNA fragment prepared as above was digested with pT7-Bl
ue vector (Novagen) with Takaraligation kit Ver.1
(Takara Shuzo) and competent cells E.coli J
Transformation was performed using M109 (Takara Shuzo). Positive clones were selected, and after confirming that the inserted fragment was present, the cells were inoculated into 25 ml of LB medium (containing 50 μg / ml ampicillin) and cultured with shaking at 37 ° C. overnight. After collecting the culture by centrifuging the culture,
Primarily plasmid D according to the method described in the lab manual Genetic Engineering Enhancement Edition pp51-52 (Masamatsu Muramatsu, Maruzen, 1990)
NA was prepared. By this operation, about 20 μg of DNA was prepared. Using 1 μg of DNA per sample as a template, a sequencing reaction was performed using a Dye Terrninatorcyclesequencing kit (Perkin Elmer), and a DNA sequencer (373A, Applied Biosystems)
To determine the nucleotide sequence. As a result, the two gene fragments amplified by PCR match the amino acid sequence corresponding to the primer design site of PPIase, and show homology with FKBP type PPIase of other organisms. It turned out to be part of the gene.

Example 5 Preparation of DAM2095 Strain Genomic DNA Library 25 μg of 30 μg of genomic DNA prepared according to Example 2
Cleavage was performed by adding a unit restriction enzyme BamHI and reacting at 37 ° C. for 24 hours. The solution after the cleavage reaction was subjected to phenol treatment and ethanol precipitation to obtain DNA. On the other hand, Ba
mHI and dephosphorylated pUC18 were purchased from Pharmacia, and a genomic DNA fragment was
Ligation kit Ver.1 was used and transformed with Ecoli JM109 to obtain a genomic DNA library of DSM2095 strain.

Example 6 Selection of Recombinant Clones Containing PPIase Gene A 270 bp DNA fragment (PN-F
1 and 1 μg of PF-R2 as primers)
The probe was labeled with random primers using Alabelling and detection kit (Boehringer Mannheim) to obtain a probe. On the other hand, a transformant prepared according to Example 4 was placed on an LB plate (including ampicillin and X-gal).
After overnight culturing, the cells were fixed on nylon membrane Hybond N (Amersham), colony hybridization was performed at 63 ° C, and washing was performed at 60 ° C.
From about 3700 clones, three colonies binding to the probe were obtained. As a result of examining the plasmid-inserted DNA fragments of these colonies, all three clones had P
It was expected to include the full length PIase gene.
Then, a sequencing reaction was performed using a Dye Terminator cycle sequencing kit, and the nucleotide sequence was determined using a 373A DNA sequencer. The resulting gene contained the full length PPIase gene as shown in FIG. The positions of the translation initiation codon and valine were estimated from the amino acid sequence and the gene sequence determined in Example 1.

[0026]

Industrial Applicability The present invention provides a thermostable FKBP-type PPIase gene that binds to the immunosuppressant FK506, 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: 527 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: Genomic DNA Origin Organism name: Methanococcus thermolithotrophicus sequence

SEQ ID NO: 2 Sequence length: 154 Sequence type: amino acid Topology: unknown Sequence type: protein Origin Organism name: Methanococcus thermolithotrophicus sequence Val Ile Phe Leu Val Asp Lys Gly Val Lys Ile Lys Val Asp Tyr Ile Gly Lys Leu Glu Ser Gly Asp Val Phe Asp Thr Ser Ile Glu Glu Val Ala Lys Glu Ala Gly Ile Tyr Ala Pro Asp Arg Glu Tyr Glu Pro Leu Glu Phe Val Val Gly Glu Gly Gln Leu Ile Gln Gly Phe Glu Glu Ala Val Leu Asp Met Glu Val Gly Asp Glu Lys Thr Val Lys Ile Pro Ala Glu Lys Ala Tyr Gly Asn Arg Asn Glu Met Leu Ile Gln Lys Ile Pro Arg Asp Ala Phe Lys Glu Ala Asp Phe Glu Pro Glu Glu Gly Met Val Ile Leu Ala Glu Gly Ile Pro Ala Thr Ile Thr Glu Val Thr Asp Asn Glu Val Thr Leu Asp Phe Asn His Glu Leu Ala Gly Lys Asp Leu Val Phe Thr Ile Lys Ile Ile Glu Val Val Glu

[Brief description of the drawings]

FIG. 1 is a photograph showing SDS polyacrylamide gel electrophoresis of purified PPIase.

FIG. 2 is a diagram showing the relationship between PPIase activity and FK506 concentration.

FIG. 3 is a diagram showing the relationship between PPIase activity and heating time.

FIG. 4 is a diagram showing a DNA base sequence of a PPIase gene and an amino acid sequence encoding the same.

[Procedure amendment]

[Submission date] June 17, 1996

[Procedure amendment 1]

[Document name to be amended] Drawing

[Correction target item name] Fig. 1

[Correction method] Change

[Correction contents]

FIG.

Claims (2)

[Claims] 1. A DNA base sequence represented by SEQ ID NO: 1,
Or a PPIase gene having a DNA base sequence substantially identical to the DNA base sequence represented by SEQ ID NO: 1. 2. A PPIase having an amino acid sequence represented by SEQ ID NO: 2 or an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 2.