JPS62248489A - Recombinant dna and production of polypeptide using same - Google Patents

Abstract

PURPOSE:To produce a polypeptide by using a recombinant DNA at least containing a DNA wherein the 3'-terminal of DNA coding Phe-Arg is bonded to 5'-terminal of a gene coding polypeptide and introducing the recombinant DNA in a microorganism. CONSTITUTION:The 3'-terminal of a DNA coding Phe-Arg is linked to the 5'-terminal of a gene coding a polypeptide using T4 DNA ligase, etc., to obtain a recombinant DNA. The DNA is introduced into a microorganism such as E.coli, Bacillus subtilis, Saccharomyces, etc., and the microorganism is cultured e.g. in a liquid medium. The obtained fused polypeptide is hydrolyzed with kallikrein to but the chain between Arg and the N-terminal of the polypeptide to obtain a polypeptide having uniform N-terminal. When the gene coding said polypeptide is devoid of Phe-Arg sequence, the gene is used as it is and when the gene contains the above sequence, it is used after converting to a gene sequence having different amino acid sequence.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to recombinant DNA and a method for producing polypeptides using the same. By utilizing the present invention, polypeptides having uniform amino acids at the N-terminus can be produced, and the resulting polypeptides are suitable for application to medicines and the like.

Conventional technology When producing 41-nogutide by expressing an animal gene, etc. using Escherichia coli using recombinant DNA technology, the N-terminus of the produced polypeptide may be methionine or formylmethionine, or may lack these. Of course, it's not the same.

Therefore, these polypeptides sometimes become an obstacle when used in medicines and the like. An example of a method for solving this problem is a method for obtaining human β-globin (European Patent Application No. 0161937). In this method, a gene encoding Els-Glu-Gly-Arg is connected to the 3' end of a gene encoding 31 amino acids from the 5' end, and this 3' end is connected to the 5' end of the human β-globin gene.
By connecting the 'ends and placing the nine genes in Grasmid of Escherichia coli and culturing this, a fused β-globin protein corresponding to the above gene was obtained, and blood coagulation factor X was applied to it. A method for obtaining a β-globin protein with a uniform N-terminus by hydrolyzing the β-globin protein is described.

Problems to be Solved by the Invention Under these circumstances, this invention solves the problem of recombinant DNA.
In the method for producing a polypeptide using the method, poIJ having a uniform N-terminus,! The present invention aims to provide a new recombinant DNA and polypeptide production method for obtaining '7'tide.

The inventors conducted research to solve the above problems,
This led to the acquisition of the following invention.

(1) 3' of DNA encoding Phs-Arg
- a recombinant DNA having at least one DNA whose terminus is linked to the 5'-end of a gene encoding a polypeptide;
0(2) 3' of DNA encoding Phe-Arg
- a recombinant DNA having at least one DNA whose terminus is linked to the 5'-end of a gene encoding a polypeptide;
A method for producing polytetraptide, which comprises culturing an organism and hydrolyzing the obtained fusion polypeptide using kallikrein.

Specifically, the DNA encoding Phe-Arg is Phe-Arg.
ha KaTTT, TTC, Arg 75E CGT
, CGC, CGA.

Since they are CGG, AGA, and AGG, all 912 combinations of these are included.

As the gene encoding poly-2ftide, genes that do not contain a Phe-Arg sequence in the molecule, such as genes for interferon r, interferon α, insulin, renin, etc., can be used as they are. Also, P in the molecule
Even if the he-Arg sequence exists, it is possible to convert the gene sequence corresponding to this sequence so that the amino acid sequence is different and use it.

To link the 3'-end of the DNA encoding Phe-Arg to the 5'-end of the gene encoding the polypeptide, a commonly known method such as T4 DNA can be used.
Use ligase.

The microorganism into which the recombinant DNA thus obtained is introduced is
Microorganisms that are commonly genetically modified include Escherichia coli, Bacillus subtilis, Satucharomyces, and the like.

A microorganism into which recombinant DNA has been introduced can be cultured by a commonly known method, for example, by inoculating the microorganism into a liquid medium and culturing the microorganism under aeration and stirring.

It is necessary to encode a methionine codon, which is a protein synthesis initiation codon, on the five-terminal side of the DNA encoding Phe-Arg.
By further inserting DNA encoding a peptide containing a large amount of acidic or basic amino acids, hydrophobic amino acids, or hydrophilic amino acids in the middle of the g codon, three types of unreacted peptides and generated peptides after the cleavage reaction with sicicalifrein can be obtained. Furthermore, it is easy to separate peptides, and it is also possible to use only about 5 to 50 single amino acids, or
Inserting DNA encoding different peptides with different amino acids facilitates the separation of the three peptides.

The obtained fusion polypeptide contains ph・-Arg in the molecule.
Since it has a sequence, when it is hydrolyzed using kallikrein, it is cleaved between Arg and the N-end of the polypeptide, yielding a polypeptide with a uniform N-end.

For hydrolysis using kallikrein, the molar ratio of kallikrein to the fusion polypeptide as a substrate is approximately 1:100 to 500, the reaction temperature is 20 to 50°C, the pH is 6 to 10, preferably 35 to 500. 4
The reaction is carried out at 0° C., p) 17.5 to 8.5.

Example 1 (Creation of a bacterium that produces rL-2 (K-H, -2) with a Phe-Arg structure added to the N-terminus of the 111L-2 protein) Using pBR322 as a vector, fL- equipped with a triggromotor 2 expression plasmid (pT9-11
) (see Figure 1). Furthermore, pT9-11
was created as follows.

First, PIE, -2-5OA (European Patent Application Publication 91
539) P+! tl, Dra), and this fragment was combined with a BamHl linker and Pat of pBR322.
A plasmid was constructed by ligating the fragment with a large fragment of 1-EeoRl (EcoR1 site was treated with Flenow) using T4 DNA ligase. Cut this glasmid with HgjAl,
After DNA polymerase I (Flenow) treatment, BamHI
I cut it with. This fragment and pDR720 were combined with Jal and Bam.
Large Hpa l -Bam obtained by cutting and analyzing with HI
The H1 fragment and synthetic oligomer a were ligated to obtain pM[-9.

On the other hand, pBR322f: Pvun and SalI were digested, and the large Pvu11-SatI fragment was ligated with synthetic oligomer 〇 to obtain pTrpA.

Both pMI-9 and pTrpA thus obtained were cut with EaoRl and BamHI, and the TL of pMI-9 was cut with EaoRl and BamHI.
The fragment containing the -2 gene and the fragment containing the trpA terminator of pTrpA were ligated to obtain pT9-11.

A part of the structure of this plasmid (pT9-11') was transformed into fL by inserting the DNA shown in Figure 2 (3).
Grasmid pT13SNeo (Fig. 2) carrying the -2 gene was constructed.

The structural difference between pT13sNeo and pT-9-11 is the part (3) in Figure 2-1, and the nucleic acid sequence of this part is as shown in Figure 2-2.

PT13SNeo plasmid, call HBIO
Bacteria integrated into I (E, coll pT13sNco/
HBIOI) was obtained by a conventional method and cultured to prepare plasmid DNA. This grasmid is treated with the restriction enzyme Neol, and then with Xhol,
DNA fragment containing the geoR1 site cleaved with Neo I, Xho I (NC' I #Xh
ol cleaved DNA fragment) was prepared.

(2) On the other hand, two types of oligonucleotides J1 shown in Figure @3 were synthesized as insert DNA using a DNA synthesizer.

■ Mix the Neo I and Xho I cut DNA fragments with two types of synthetic oligonucleotides, and add T4O
Apply NA IJ gauze to ligage and insert Mat-Arg-Pro-P just before the 2nd protein.
Grasmid (p
T13sxtt, -2) t-obtained.

■ Obtained pT13sKTL-2 and E, coll H
Nine BIOI bodies were treated with TCM (10mM Tris-HCt
pH7.5, 10mM CaC12, 10mM MgC2
2) 200μA! Keep at 0℃ for 20 minutes, then 4
2'C, let stand for 2 minutes, and then add Ltnl Noshiichi Gloss (Trigton 1%, Yeast Processing 0.5%, N
aCL 0.5%, glucose 0.2%) was added and the mixture was shaken at 37' for 1 hour. The microbial cell sections were collected by centrifugal sedimentation, diluted appropriately with water, and ampicillin (5%) was added.
0γ//! /) was smeared on an L-70 plate containing the following. Colony 1 that appeared after culturing this plate at 37°C for 20 hours
I caught 0 bacteria.

(2) Each of the picked colonies was cultured on a single ion plate, the cells were collected, and siblasmid DNA was prepared by a conventional method. One strain (E
, eol I, pT-13SK IL-2/HB
IOI) was selected.

(2) Cultivation and product acquisition Selected p'r 13SK IL-2/HB 101
, trizotone 1%, yeast extract 0.5%, Na
Shaking culture was carried out at 31° C. for 16 hours using a Sakaro flask containing a medium IQQa/ having a composition of Cj O, 5fb and Gycose 0.2 parts.

Culture solution 83- was mixed with 1.51 M9-casamino acid medium (cabuminic acid 1.0, yeast extract 0.2, NH4C20,
Section 5, MgSO4-7H200,05%, C&C12-
2H200,005%, L-L@% 0.04%, L
-ProO, 04's, VBl-HCtO, OO04 arrogant,
Glucose 2 chronic, KH2PO40,1 wide.

TMA-812 (antifoam) 0.02, ampicillin 100 All/'rL1. streptomycin 25p
The mixture was inoculated into a 31-volume mini-Japanese tanker filled with 70 Q rprn of water (containing l/rug) under aeration and agitation conditions of 70 Q rprn and soybean m.

Control culture was carried out at 31°C, p) 16.2. 25 when the bacterial cell turbidity (OD66onm) grows to 4.5.
till/ml O3-iatol acrylic acid and feed medium (casamino acids 1%, L-Le710.04%
, L-Pr.

0.04%, VB-HCA 0.0004'j
, TMA-8120,00125ml? /dt was added, and the culture was further continued for 10 to 20 hours.

The granules produced inside the bacterial cells were extracted using the following procedure, the oral bacterial cell concentration was concentrated twice by centrifugation, and Lysochy A O,
0411/l, after adding EDTA 015M,
The mixture was stirred at 5 to 15°C for 3 to 6 hours, and then the bacterial cells were destroyed by ultrasonic disruption, and the granules were collected by centrifugation at 8000 rprn*5 min.

This solution, K-rL-24 degrees is 100 pH/d,
The concentration of the solution was adjusted to 2M guanidine, 1mM of oxidized glutathione and 10mM of reduced glutathione were added thereto, and the mixture was inked and incubated at room temperature for 10 to 16 hours at PHs, o. Next, this solution was concentrated with a holofiber and 5epadaxG-25
Guanidine is removed by filtration with l''k(p).

Furthermore, ion exchange chromatography using CM-S@pharoae was performed, and then 5ephade
A fraction corresponding to K-IL-2 was obtained by filtration with xG-25.

As a result of testing the amino acid sequence of the N-terminal side of this substance using protein sequencing, it was found that M@t-Arg-P
ro -Pha -Arg -rL-2 (KJL-2)
It was confirmed that

(3) 50mM Trim- containing 113mMNact cleaved by kallikrein
In HC1 buffer, pH 7,8, obtained 9 to 1 L-2
After reacting 825~ with human plasma kallikrein at 37°C for 16 hours, a fraction corresponding to Leu 2 was fractionated by reverse phase HPLC.

As a result of analyzing the amino acid sequence near the N-terminus using protein sequencing, it was confirmed that K-rL-2 was quantitatively converted to Ru2 protein.

In addition, even if unconverted -IL-2 remains in the microstructure, K-TL-2 and fL-2 can be completely separated by FPLC ion conversion chromatography.

Example 2 (1) Phs-Arg on the N-terminal side of TL-1β protein
Construction of a butterfly that produces fL-1β (fL-1β-K) with an added structure■ Using pBR322 as a vector, an IL-1β expression plasmid (pT
Plasmid p'r was obtained by converting the IL-2 gene portion of Grasmid into the IL-1β gene using 9-11).
TL-1β (Figure 4) was constructed.

The structural difference between p'r tt, -1β and p'r 9-11 is the part (3) in Figure 4-1, and the nucleotide sequence of this part is shown in Figure 4-2. It is a communication. pTIL-
A bacterium in which the 1β plasmid was integrated into E. coli HBIOI (E. callp'r tt, -1β/HBIO
I) was obtained by a conventional method and cultured to prepare plasmid DNA. Restriction enzyme C1m is added to this plasmid.
1 and then Hl nd■ to form -C1
a I, DNA fragment cut with Hlndll and containing nine EeoR7 sites (C1a l.

) (ind [1 cleavage DNA fragment) was prepared.

(2) On the other hand, two types of oligonucleotides b shown in FIG. 5 were synthesized as insert DNA using a DNA synthesizer.

■C1ml, Hlndill cut DNA fragment and two types of synthetic oligonucleotides were mixed, and T
4 DNA IJ gauze was applied to perform lidarcining, and M@t-Asp- was added just before the TL-1β protein.
Asp -Asp -Val-Aap -Phe -A
A plasmid with a structure encoding rg (p'r tt
, -1β-K) were obtained.

■ The obtained p'r rL-1β-K was transfected into E. coll HBIOI bacterial cells in the same manner as in Example 1, and the cells were cultured on a broth plate containing Nadon Picillin (50 r/rR1). Twenty colonies were harvested.

(2) Each of the picked colonies was cultured on a bouillon plate, the cells were collected, and siblasmid DNA was prepared by a conventional method.

This grasmid was treated with the restriction enzyme Sal I, and one strain was selected from among the strains in which cleavage of grasmid by this enzyme was confirmed.
Strain (E, colt, pT II, -1β-/H
BIOI') was selected.

(2) Culture and product acquisition by the same method as Example 1 F) E, coll pTT
The protein produced by L-1β-/HBIOI is
-Asp -Asp -Asp-Val -Asp -
It was confirmed that it was Phs-Arg-IL-1β.

(3) Cutting with kallikrein As a result of performing the same method as in Example 1, TL-1β-K
was quantitatively converted to TI, -1β protein.

[Brief explanation of drawings]

FIG. 1 is an explanatory diagram of the production progress of TL-2 expressing grasmid pT9-11. Figure 2 shows plasmid PT13 containing the TL-2 gene.
It is an explanatory diagram of 8Nco. (1) is DNA derived from pBR322. (2) is DNA derived from pT9-11. (3) is the structurally converted DNA portion of pT9-11 (shown in Figure 2-2). FIG. 3 is an explanatory diagram of synthetic oligonucleotide a. ! Figure 4 is an explanatory diagram of the plasmid p'r rt, -1β containing the converted TL-2 gene. (1) is DNA derived from pBR322. (2) is pT9- 11. (3) is the DNA part whose structure has been changed from pT9-11 (shown in Figure 4-2). Figure 5 shows the synthetic oligo9 nucleotide b FIG.

Claims (2)

[Claims] (1) A recombinant DNA having at least a DNA in which the 3'-end of the DNA encoding Phe-Arg is linked to the 5'-end of the gene encoding the polypeptide. (2) A recombinant DNA having at least a DNA in which the 3'-end of the DNA encoding Phe-Arg is linked to the 5'-end of the gene encoding a polypeptide has been introduced. A method for producing a polypeptide, which comprises culturing a microorganism and hydrolyzing the obtained fusion polypeptide using kallikrein.