JPS633790A - Recombinant plasmid - Google Patents

Abstract

PURPOSE:A plasmid recombinant producing protein showing epidermal growth factor (EGF), efficiently, having a structural gene to code a specific polypeptide inserted into the plasmid recombinant. CONSTITUTION:A DNA base sequence of a structual gene of protein factor (EGF) having epidermal growth activity and a translation control signal range is synthesized to give ECF gene (A) having a recognition breakage site of restric tion enzyme XbaI between a gene codon at the top of the structural gene and SD sequence. Then manifestation plasmid vector pDR540 having tac promoter is scissored with BamHI to give cleft cloning vector (B). The components A and B are linked with ligase T4 to give recombinant plasmid pPGFLI (C). Then the component C is transduced into Escherichia coli JM103, the prepared transformant is cultivated and multiplied. Then the multiplied mold is extracted and separated to give a recombinant plasmid (figure) into which a structure gene to code a polypeptide consisting of 53 amino acids having amino acid in the downstream of the tac promoter is inserted.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to a method for synthesizing a protein having an epidermal proliferation effect using genetic engineering techniques, and in particular to a plasmid suitable for searching for and studying conditions for highly efficient production thereof. Regarding recombinants.

[Conventional technology]

Generally, when using genetic engineering techniques to cause microorganisms to produce proteins that they do not originally produce, the following three factors must be satisfied in order to improve the synthesis efficiency. These are (1) use of cloning vectors with high transcription and translation efficiency, (2) selection of host (microorganism) systems that effectively express the characteristics of the vector, and (3) production of proteins from microbial culture fluid. Establishment of a highly efficient collection method for
It is. Among these, the one with the greatest effect is (1)
This is a factor.

To date, there have been four reports regarding the production of a protein factor with epidermal proliferation activity (hereinafter abbreviated as EGF) using genetic engineering methods (for example, JP-A-57-1'209
Publication No. 6). Both are natural W consisting of 53 amino acids.
BGF6 or BGF6 is a self-produced fusion protein in which a peptide chain consisting of several or more amino acids is linked to its N-terminus. In all of these reports, a plasmid in which the structural gene of EGF is linked to a plasmid vector containing a gene expression promoter that is known to have relatively high transcription efficiency among gene expression promoters commonly found in E. coli, yeast, etc. We are trying to produce proteins using recombinants.

The structural gene of EGF is designed based on the amino sequence information of natural EGF and synthesized organically. At this time, gene codons are selected depending on the host so that the translation efficiency within the microorganism is not reduced. That is, when gene codons are degenerate for one amino acid, codons that appear frequently in E. coli or enzymes are selected.

However, these reports have focused on a translational control signal that is clearly a trap that has a significant impact on translation efficiency, namely the distance between the first codon (ATG) of a structural gene and the SD sequence immediately preceding it. Kata, Imamoto, Proteins/Nucleic Acids/Enzymes, vot, 28°, Kai 14.pp, 28-4
7. (1983)) are not considered at all.

[Problem that the invention seeks to solve]

The purpose of the present invention is to have the ability to produce EGFt in E. coli, and to easily enzymatically change the interval between two translation signals (SD sequence and translation start codon). The purpose of the present invention is to provide a plasmid recombinant: pEGFLI, which enables the investigation of conditions for the highly efficient production of EGF (53 amino acids).

[Means for solving problems]

There are two methods for obtaining the structural gene DNA that carries the sequence information of EGF, which consists of 53 amino acids. (1
) Cells that actually produce EGF) Separate and purify the messengers R and NA for EGF using the hybrid DNA pairing method.
9DNAt-biochemically synthesized and (2) E
Among the several gene base sequences predicted from the amino acid sequence of GF, the gene codons used therein are used in the host.

After considering whether it appears frequently and whether it has a structure that is unlikely to cause mistakes during organic synthesis, select one base sequence that seems to be the most appropriate, and use it in organic chemistry. Sometimes they are synthesized. It is easier to synthesize 159 base pairs corresponding to 53 amino acids using organic chemistry.
In the present invention, method (2) was selected because it is much easier to search for 13×10” base pairs (base pairs estimated to exist in seven cells).

The existing tac promoter was used as a promoter to express the synthesized gene.

Furthermore, in order to make the interval between the above two translation control signals variable, in the present invention, a recognition switching site for a restriction enzyme that exists only here in the entire recombinant plasmid is inserted between the two signals. did. Therefore, in the present invention,
Not only the structural gene of EGF but also the DNA of the translation control signal region were chemically synthesized.

[Effect]

As a result, the recombinant plasmid was cut, repaired, and ligated with the enzyme, thereby making it possible to make the interval between the two signals described above variable.

〔Example〕

An example of the production of plasmid recombinant pEGFL 1 will be described below.

First, we will describe the DNA sequence design and synthesis of the EGF structural gene and translational control signal region. FIG. 2 shows the amino acid sequence of EGF and the corresponding gene. E
Since the amino acid sequence of GF was determined by Qregory et al., its structural gene can be predicted from the amino acid/gene codon correspondence table. However, this correspondence is not unambiguous because there is some duplication on the gene codon side. Therefore, we created a host-specific gene codon frequency table (Ikemura, Cell Engineering).

vot, 2,413. pp, 78-89 (198
3) With reference to), gene codons that appear frequently in E. coli were selected for each amino acid, and 159 genes consisting only of them were selected.
First, we designed a structural gene blueprint for bases.

Furthermore, we placed an SD sequence (AGGA) t- upstream of the translation start codon ATG; separated by 8 bases, and created a recognition/cleavage site for the restriction enzyme XbaI in the center of both signals to design a double-stranded DNA with a total length of 185 base pairs. did.

However, a series of DNA chain elongation reactions using the phosphotriester method, which is a known DNA synthesis method, cannot synthesize long-chain DNA as shown in FIG. 2 all at once. That is, even if the yield of the elongation reaction for one base is 90%, if the reaction is repeated 20 times, the total yield will be about 12%, making purification difficult. Therefore, in reality, the long double-stranded DNA in Figure 1 is converted into a block of 28 single-stranded oligonucleotides (A-5-1 to A-14, B-
1 knee B-14), and each was synthesized by a DNA chain elongation reaction using the phosphotriester method.

The reason for this blocking is that when these blocks are put together to create the EGF gene, inappropriate interactions may occur between the blocks, resulting in the formation of a gene with an incorrect structure. The base sequence of each block was optimized to avoid

FIG. 4 shows a process in which a mononucleotide (a) bonded to a polystyrene resin is subjected to condensation polymerization with another mononucleotide (b) to synthesize a dinucleotide (C).

In order to synthesize an oligonucleotide consisting of n nucleotides, this step may be repeated (n-1) times.

Details of the reaction conditions are detailed in the cited references cited above.

FIG. 5 shows the procedure for ligating blocked oligonucleotides using T4 ligase. Since there was a risk that an incorrect combination would occur if the 287° locks were connected all at once, a two-step connection method was adopted. The reaction followed a conventional method.

Next, as shown in FIG. 1, the procedure for molecular cloning of the synthesized EGF gene into an expression vector is shown.

First, we used the β-lactamase gene (Amp') and the galactokinase gene (Qalk) as selectable marker genes, and further used the existing tac promoter, which is a fusion of the 35 region of the tryptophan promoter and the pribno niece sequence of the lactose promoter. Expression plasmid vector pD 540(a) was digested with restriction enzyme BamHI.
It was cut and cleaved. This was then treated with bacterial alkaline phosphatase to obtain a cleavage cloning vector (b) fi- whose I terminus was dephosphorylated.

Furthermore, the above-mentioned synthetic EGF genes (C) and (b) were ligated using a ligating enzyme T4 ligase to obtain a novel recombinant plasmid pEGFL1 (d). E. coli JM103, which was used as a recipient cell, was then transformed with plasmid (d) using the rubidium-calcium method. This bacterium was cultured on an agar plate medium containing ampicillin, and the resulting colonies (containing the ampicillin resistance gene A m p', i.e. p
EGFLl-containing bacteria) were isolated.

After culturing and proliferating this in a liquid medium, the recombinant plasmid vector (ψ) was recovered from the bacterial cells (Molecul
ar Cloning, ed by Maniati
s et al.

Co1d 3prHng 1 (arbor Labor
atory, pp.

(1982)).

FIG. 1(e) shows the restriction enzyme map of (d).

〔Effect of the invention〕

Plasmid vector pEGFL produced according to the present invention
1, the recognition cleavage site for the restriction enzyme , the interval between the two signals can be changed by enzymatic treatment. FIG. 6 shows a method for producing pEGFL1 variants with various spacings.

In pEGFLl(a), the interval between the SD sequence and the ATG:ffton is 8 bases (see Figure 2). (a) to Xba
Cleavage with I yields (b), which is treated with 81 nuclease to blunt the cleavage site (f), and then ligated with Ta liger I, resulting in a distance of 4 bases between the two signals. pEGFLl (modified position) can be exploded.

On the other hand, [lenow 72] of DNA polymerase is added to Φ).
When treated with a compound, the single-stranded portion at the cut end becomes double-stranded, yielding (C). Next, by ligating this with T4 ligase, the distance between the two signals becomes 12 bases.
Obtain GFL1 variant (d). At this time, a new recognition cleavage site for the restriction enzyme Alu is generated. Therefore, newly (d)
(e) is obtained by cutting with A l u I, and (i) can be produced by ligating it with the HapI linker (), but after cutting this with the restriction enzyme Hapl, this Similar to the procedure described above, S1 nuclease and DN
By applying A polymerase, the distance between the two signals is 14 bases and 18 bases, respectively.
2 variants), (A can be obtained.

In this way, five types of modified versions of the original recombinant plasmid vector 1) EGPLI (a), d-, etc., in which the distance between the two signals is 4, 8, 12, 14, and 18 bases, are obtained by operations using only enzyme reactions. be able to.

During the above process, when 81 nuclease is allowed to act,
By varying the reaction conditions such as the concentration and quantitative ratio of the enzyme and substrate, and then by allowing DNA polymerase to act on the reaction product, the size of the DNA end to be blunted can be adjusted. interval 5-7 bases, 13
It is also possible to create base variants.

By introducing the various pEGFL1 variants prepared in this manner into bacterial cells, culturing the host under various conditions, and expressing BGF, the interval between the two signals with the highest translation efficiency can be found. The knowledge gained here is versatile! It can also be applied to cases where a structural gene other than EGF is incorporated and its expression is attempted.

[Brief explanation of the drawing]

Figure 1 is an explanatory diagram showing the preparation of a recombinant cloning vector according to the present invention and restriction enzyme recognition sites, Figure 2 is a diagram showing the entire synthesized base sequence, and Figure 3 is a diagram showing the entire base sequence with 28 synthetic oligos. Diagram divided into nucleotide blocks, No. 4
FIG. 5 is a diagram showing the flow of oligonucleotide synthesis, FIG. 5 is a diagram showing the flow of assembly and connection of synthetic oligonucleotide blocks, and FIG. 6 is an explanatory diagram showing an application example of the present invention. A...adenine, C...cytosine, G...guanine, T...thymine, SD sequence -3hine, ])
a1garnow sequence, A-1 to 14, B-1 to 14・
・Synthesized DNA fragment 2nd part 30

Claims (1)

[Claims] 1. 53 having an epidermal proliferation effect downstream of the tac promoter
1. A recombinant plasmid, characterized in that a structural gene encoding a polypeptide consisting of 5 amino acids has been inserted. 2. In the plasmid recombinant according to claim 1, a restriction enzyme recognition cleavage site is provided between the first gene codon of the structural gene and the SD sequence, and the interval between the two is modified using an enzyme. A recombinant plasmid, which facilitates examination of gene translation efficiency. 3. The plasmid recombinant according to claim 2, which has a recognition cleavage site for the restriction enzyme Xba I between the first gene codon of the structural gene and the SD sequence. body.