JPH0213381A - Vector for expressing plural proteins and preparation of proteins using the same vector - Google Patents

Abstract

NEW MATERIAL:A vector capable of being multiplied in a predetermined host and having at least an SD arrangement, a translation initiating codon, a structural gene and a termination codon or the SD arrangement, the translation initiating codon, a signal peptide, the structural gene and the termination codon under controlling one promoter or individual promoters. USE:A vector capable of efficiently prepare plural proteins at once by a genetic engineering means. PREPARATION:For example, a region required for expressing proteins by a genetic engineering means is combined with a known vector (e.g., pTA2622R) and a structural gene for one or more kinds of the objective proteins (e.g., hEGF and hTGF) is combined with the vector at a specific site of the vector to provide the vector having an SD arrangement, a translation initiating codon, a signal peptide, the structural gene, a termination gene, etc., and expressing plural proteins.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel vector, a transformed specimen containing the same, and a method for producing a plurality of desired proteins using the same.

In recent years, the technology of using genetic engineering techniques to make microorganisms produce peptides or proteins (hereinafter referred to as proteins, etc.) has become commonplace.

Currently, various improvements are being made to improve production efficiency when producing proteins and the like using this technology.

Production of proteins, etc. by genetic engineering means that a DNA gene corresponding to a desired protein, etc. is inserted into a vector to produce recombinant DNA, and then this is integrated into a host microorganism to transform the host and create a transformant. obtain. Next, the desired protein is produced by culturing it under appropriate conditions, and then the desired protein and the like are recovered.

In such a process, improvements aimed at improving production efficiency include, for example, improvements in host bacteria and vectors, and improvements in methods for collecting and purifying proteins and the like.

Among these, various improvements have been proposed regarding vectors. The vector here refers to the gene of the target protein, etc. (hereinafter also referred to as "foreign gene")
It is a carrier DNA that has the function of introducing, proliferating, and expressing a host bacterium. The vector usually contains a promoter, an operator, a transcription initiation region, a liposome binding region (RBS), etc. to carry out this function.

However, vectors can be improved by replacing them with those with strong promoter activity (tryptophan promoter, PL promoter, etc.), or by replacing them with those that have DNA regions that have traditionally been involved in high expression of proteins (for example, riboprotein-derived vectors). Things [EMBOJournal, 3.243
7-2442 (1984)]) transcription initiation region or R
High expression can be achieved by using BS, etc., by appropriately combining with other promoters, operators, or RBS of different origins, by improving the 3° untranslated region, or by introducing a runaway type plasmid. Attempts are currently being made to create vectors.

However, at present, the combination of each region does not work well, and unlike the riboprotein-derived DNA region, there is no clear operator, making it difficult to arbitrarily regulate and control gene expression. As it is, there are problems such as difficulty in using it from the viewpoint of genetic recombination technology.

Therefore, at present, there are not many vectors that can consistently achieve high expression and recovery of various foreign genes.

The present invention was developed through intensive research to develop vectors useful for producing proteins, etc. using genetic engineering techniques. If one of these is capable of secreting a desired protein outside the bacterial cell and/or into the cell plasma, and the other is capable of expressing and recovering a different desired protein within the bacterial cell, then they will be able to interact with each other. This new finding was based on the discovery that different desired proteins can be expressed simultaneously and recovered separately.

Therefore, the vector of the present invention must be able to propagate in the intended host and have at least the following translation units (1) and (2) under the control of one promoter or under the control of separate promoters. This is a characteristic feature.

(1) A) SD sequence, mouth) translation start codon, c) structural gene, 2) termination codon (2) a) SD sequence, mouth) translation start codon, c) signal peptide, 2) structural gene, e) termination Codon In addition, the protein production according to the present invention is characterized in that it consists of the following steps a) to c).

b) Prepare a vector that is capable of propagating in the intended host and has at least the following translation units (1) and (2) under the control of one promoter or under the control of separate promoters.

(1) A) SD sequence, mouth) translation initiation codon, c) structural gene, 2) termination codon (2) a) SD sequence, mouth) I! Translation start codon, c) signal peptide, d) structural gene, phantom stop codon) Transform a host cell using this vector to prepare a transformant.

c) Cultivating this transformant and recovering the produced desired protein.

The vector of the present invention is capable of expressing a plurality of desired proteins at the same time, and is capable of recovering these proteins separately from inside and outside the bacterial cell and/or from the periplasm, thereby enabling efficient substance production.

Vector In the present invention, a vector refers to a vector that is capable of propagating within the intended host and that contains at least the following translation units (
1) and (2).

(1) SD sequence, mouth) translation initiation codon, c) structural gene, 2) termination codon (2) a) SD sequence, mouth) B translation initiation codon, c) signal peptide, 2) structural gene, phantom termination Codons and, if necessary, regions involved in increasing gene transcription efficiency, regions or base sequences that increase translation initiation efficiency of structural genes, 8' untranslated regions that contribute to mRNA stabilization, and plasmid copy number. It may contain or be deleted, such as a region involved in . Such vectors can be prepared based on conventional genetic engineering techniques. For example, based on known vectors, the necessary regions can be chemically synthesized or obtained from other vectors. This can be easily accomplished by appropriately integrating the region into the vector.

For example, the Trp promoter gene is present on the chromosome of E. coli, and therefore can be easily obtained by cloning the promoter from the chromosomal DNA of E. coli according to a conventional method. Here, the promoter region refers to a region to which RNA polymerase, an enzyme that transcribes genetic information from DNA to RNA, first binds. The region having the function of secretion to the outside of the bacterial cell and/or to the heliblasm has a translation unit consisting of a) an SD sequence, an) translation initiation codon, c) a signal peptide, b) a structural gene, and e) a termination codon. In particular, signal peptides include those that allow the structural gene of a desired protein to be bound immediately after the downstream end of the gene encoding the signal peptide.For details, see Japanese Patent Application Laid-Open No. 37099/1983.

On the other hand, the region in which a desired protein can be expressed and recovered in a host cell has a translation unit consisting of a) an SD sequence, a) a translation initiation codon, c) a structural gene, and b) a termination codon. It differs from the above-mentioned translation unit with secretion function in that it does not have a signal peptide. That is,
This is a so-called fusion method in which the structural gene of another desired protein is directly linked downstream of the translation initiation codon via a linkage consisting of a cleavable amino acid sequence, or a direct linkage method in which the desired structural gene is linked downstream of the translation initiation codon. , these are all known methods.

Vector 1 The above-mentioned recombinant vector is used in the production process of the target substance according to a given genetic engineering method after integrating one or more target structural genes such as proteins into a specific position. (See Examples below for details). Specifically, insulin, interferon (α, β), interleukin (I to IV), calstonin, EGF%GH, NGF, TGF (α, β),
Any substance can be expressed, such as EPO and IGF (1, ■).

The expressed desired protein can be separately recovered from within or outside the bacterial cell and/or from the hemoplasm by a known method.

For detailed recovery methods, please refer to 1) Direct expression method: JP-A-8-134998, JP-A-60-28994, 2) Fusion method: JP-A-62-304,937, JP-A-63. -41615 specification, 3) Secretion method: JP-A-61-
152297, JP 61-1-2278, JP 61-152279, JP 61-181396,
JP-A-61-254196, JP-A-61-28029
No. 2, JP-A-62-158491, JP-A-63-37
No. 99, JP-A-63-3800, JP-A-63-744
No. 85 and Japanese Patent Application No. 44585/1985, Japanese Patent Application No. 1987-
Please refer to the specification of No. 309858.

1) Preparation of hBGF and hTGF/TGFα 1, pT
Preparation of A2632R pTA2622R (see Japanese Patent Application No. 62-44585) 10Rg was mixed with 6mM Trls-HCI (pH 7,
9) A linear chain was formed by reacting in 5all of 3 units at 87°C for 2 hours in 50Rg of 150mM NaCl, 6mM MgCl□ solution.

After phenol treatment and ethanol precipitation, it was dissolved in 50 μm of the above solution and partially cleaved with 1 unit of EcoRI.

Thereafter, the fragment (1) containing the Trp promoter and Km (kanamycin) resistance gene was obtained by agarose electrophoresis.

5 μg of pTA1632 (see JP-A-61-149087) was cut with 8 units of BcoRI and 5all in 50 μe of the above solution to obtain bla signal-hEG.
A fragment of approximately 220 bp (II) containing the F gene was recovered from the agarose gel. (1) and (II
) was closed using a DNA ligation kit (Takara Shuzo), and E. coli HBIOI was transformed. A plasmid was prepared from the transformant to obtain pTA2632R (see Figure 1).

2. Creation of pTA26 017 32R pFM-TGF
10Rg (3mM Tr l 5-HCl (pH
8,0), 20mM KCl and 6mM Mg
8 un l in Cl 2 solution for 2 h at 37°C.
The reaction was carried out with S m a I and Hpal of t.

Then, by agarose gel electrophoresis, h E G
A fragment (II) containing the F/T G Fa gene was obtained (see Figure 2).

After cutting 5 μg of pTA2632R with 8 units of Hpal in the above composition solution, phenol extraction, and ethanol precipitation, it was dissolved in 50 με of IMTris-HCI (pH 8,0), and 1 unit of Bacterial Alkalfne phosphatase was added.
Phosphatase (BAP)] was purified by agarose gel electrophoresis at 660°C for 1 hour to obtain a fragment of 1 (see Figure 2).

(1) and (II) were closed using a DNA ligation kit (Takara Shuzo), and E. coli HBIOI was transformed. A plasmid was prepared from the transformant and pTA2
6-017-32R was obtained (see Figure 2). This vector has a stop sequence after hEGF/TGFα under the control of the trp promoter, and further has a bla signal-hEGF gene via an SD sequence downstream thereof.

3.3s gene expression and recovery B.coli 88101 strain containing pTA26-017-32R was prepared at 50 tt g/
L-brout 5m containing kanamycin of m e
and cultured overnight at 37°C. 50μ of this
50Rg/m J! of kanamycin, 0.2
M-9 medium containing 51% of casamino acids! , inoculated into 3
A shaking culture was performed at 7°C.

After 24 hours, the bacterial cells were collected by centrifugation, and production of hEGF/hTGFα fusion protein (a in Figure 3) was confirmed by 5DS-polyacrylamide gel electrophoresis as shown in Figure 3.

Note that the band in FIG. 3 is the band of the kanamycin resistance gene product and was not observed in pFM-TGF.

On the other hand, hEGF secreted into the culture supernatant was analyzed using IEIA (En
zyme Immuno As5ay). The table below also shows the results of culturing pFM-TGF under similar conditions (when culturing pFM-TGF/HB 101,
50 μg/me of ampicillin was added instead of kanamycin).

Quantification of hECF secreted into the medium N, D: Undetectable As a result, hBGF was detected in the bacterial cells 24 hours after the start of culture.
/hTGFα fusion protein band is clearly observed.

On the other hand, hEGF was 530 μg/ncult after 24 hours.
The amount of secretion of ure is shown.

Collecting hEGF from the culture supernatant 24 hours after the start of culture,
Purified. At the same time, bacterial cells hEGF/hTGFα
We wish to be able to recover the fusion protein of hEG.
For the method of recovering hTGFα from the F/hTGFα fusion protein, please refer to the specification of Japanese Patent Application No. 128549/1983)
.

[Brief explanation of the drawing]

FIG. 1 shows the method for producing pTA2632Ft. Figure 2 shows the hGH/hTGF fusion protein and hEGF.
! Simultaneously expressed vine vector pTA26-017-32
This shows a method for producing R. Figure 3 shows the expression of hGH/hTGF fusion protein.
It is a figure confirmed by DS-polyacrylamide gel electrophoresis.

Claims (1)

[Scope of Claims] 1. A vector that is capable of propagation in a intended host and has at least the following translation units (1) and (2) under the control of one promoter or under the control of separate promoters. (1) A) SD sequence, B) Translation start codon, C) Structural gene, D) Stop codon (2) A) SD sequence, B) Translation start codon, C) Signal peptide, D) Structural gene, E) Termination A method for producing a protein, comprising codon 2 and the following steps a) to c). b) Prepare a vector that is capable of propagating in the intended host and has at least the following translation units (1) and (2) under the control of one promoter or under the control of separate promoters. (1) A) SD sequence, B) Translation start codon, C) Structural gene, D) Stop codon (2) A) SD sequence, B) Translation start codon, C) Signal peptide, D) Structural gene, E) Termination Codonro) Transform host cells using this vector to prepare transformants. c) Cultivating this transformant and recovering the produced desired protein.