JPH01257482A - Dna having synthetic gene for producing human epidermal growth factor and plasmid recombinant thereof - Google Patents

Abstract

PURPOSE:To mass-produce a hEGF (human epidermal growth factor) monomer, by integrating a gene of a polymer hEGF factor consisting of plural human epidermal cell growth factors linked through one or more lysines in an expression vector, transforming Escherichia coli therewith, producing a polymer and separating the resultant polymer. CONSTITUTION:Any of tac promoter, trp promoter and lacUV promoter or several thereof are arranged through preferably a nucleotide expressed by formula III on the upstream side of a DNA having hEGF expressed by formula II (Pi, m is A or G; m and n are natural numbers; i is a number of >=1-<=m) when the DNA sequence expressed by formula I is assumed as SEQ to enable highly efficient production of a polypeptide in bacterial cells of Escherichia coli.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to a method for synthesizing a protein having a human epidermal proliferation effect using genetic engineering techniques, and particularly relates to a method for synthesizing a protein having a proliferative effect on human epidermis using a genetic engineering method, and particularly a method for synthesizing a protein having a synthetic gene that enables highly efficient production. Regarding the plasmid recombinant.

[Conventional technology]

In general, when genetic engineering techniques are used to cause a microorganism to produce a protein that it does not originally produce, the following three factors must be mainly satisfied in order to improve its synthesis efficiency. These are (1) the use of cloning vectors with high transcription and translation efficiency, (2) the selection of host (microorganism) systems that effectively exhibit the characteristics of the vector, and (3) the high yield of protein produced from microbial culture. Establishment of efficient collection method,
It is. Among these, the one with the greatest effect is (1)
This is a factor.

In order to achieve high-efficiency synthesis of products, the following five factors can be summarized as factors that should be considered when selecting or constructing a cloning vector to be used.

(a) Structure of transcription promoter, operator, and transcription termination nucleotide sequence.

(b) Structure of a translation control nucleotide sequence containing an SD sequence.

(c) Compatibility of gene codons defining amino acids with the host.

(d) Protection of produced proteins from degradation.

(e) Gene amount (copy number).

Human epidermal growth factor (hEGF), which can be produced with high efficiency in the present invention, is a polypeptide hormone consisting of 53 amino acids that is normally secreted mainly from the human duodenum or submandibular gland. Gregory (
H, Gregory) *Nature
, 257, 325 (1975)).

It has the effect of suppressing gastric acid secretion and promoting proliferation of epidermal cells. These physiological activities indicate that hEGF is effective against gastric ulcers.

It has shown potential as a treatment for duodenal ulcer.

It is also known that hEG acts on hEGF receptors distributed on the surface of cell membranes and promotes the expression of various biological reactions. (Dei Gospodarovit.

Manual Review of Physiology) (D,G
ospodarovicz, Ann, Rev, P
In recent years, the relationship with carcinogenic mechanisms has also attracted attention. In other words, it is known that the products of erbB-1 and B-2, which are types of oncogenes, have a high structural relationship with the hEGF receptor based on their amino acid sequences.
Elucidation of the relationship between EGF and cancer development is awaited.

However, since naturally occurring hEGF is extremely small, mass production through genetic engineering has attracted attention.

There have been 15 reports so far regarding the production of hEGF by genetic engineering methods (for example, Japanese Patent Application Laid-open No. 1161
-8881 publication).

In all of these reports, a plasmid set was created in which the hEGF structural gene was linked to a plasmid vector containing a gene expression promoter commonly found in Escherichia coli, yeast, etc. that is known to have relatively high transcription efficiency. Attempts are being made to produce proteins using modified organisms (measures against factor (a) above).

The structural gene of hEGF is designed based on the amino acid sequence information of natural hEGF 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, if the genetic codons are degenerate for one amino acid, codons that appear frequently in E. coli or enzymes are selected (measures against factor (C)).

As a response to factor (b), there are many examples of using one of the several types of SD sequence regions originally held by E. coli.

Furthermore, foreign proteins with relatively small molecular weights, such as hEGF, are unstable within the bacterial cells because they are easily targeted by proteolytic enzymes. Therefore, as a countermeasure for factor (d), the gene for the exocrine secretion signal peptide is linked to the upstream side of the hEGF gene to promptly secrete the produced protein to the outside of the bacterial cell and protect it from proteolytic enzymes. The use of hosts with low production capacity (Kanmoto, Protein Nucleic Acid Enzyme, Wangyo, Nα11°1039 (1986)) is being carried out.

As a response to factor (a), there are many examples of using a relaxed plasmid that has a Co1EI-based plasmid replication origin (or i) and has a large intracellular copy number.

In addition, personnel (Bio Industry (Bi.

+Industry), 3. Nal 2.875
(1986)), there is also an example of using a recombinant vector having a set of multiple gene expression control sequences and hEGF structural gene in one plasmid vector molecule.

[Problem that the invention seeks to solve]

However, in designing a recombinant plasmid using the above-mentioned conventional techniques, sufficient consideration was not given to the recovery, separation, and purification of the produced protein from the medium. As mentioned above, low molecular weight proteins such as hEGF are not only unstable within bacterial cells, but also require various separation means (e.g. electrophoresis, high performance liquid chromatography, etc.) and detection means (Coomassie blue staining, etc.).

This is disadvantageous in terms of silver staining, 81IS-methionine incorporation detection, etc.). Therefore, even when using a host-vector system that has highly efficient transcription signals (promoters) and translation signals and potentially has the ability to mass-produce hEGF, it is difficult to stably maintain high efficiency productivity. Ta.

The purpose of the present invention is to incorporate a multimeric hEGF gene consisting of multiple hEGFs linked via one or more lysines into an expression vector, transform Escherichia coli with the gene, and make the bacterium susceptible to intracellular proteolytic action. Once a stable large molecular weight hEGF precursor is produced, it can be efficiently recovered from the culture solution by 9 separations.

To provide a means for efficiently producing hEGF by purifying it and then treating it with a protein hydrolase in an in vitro system to obtain a larger number of monomeric hEGF than one molecule of multimeric hEGF. It is in.

[Means for solving problems]

The above purpose is to promote hEG through the amino acid sequence arginine (lysine), which is easily cleaved by the polypeptide chain hydrolase nitrypsin, which E. coli does not originally have.
F is achieved by creating an expression vector in which a plurality of linked structural genes of multimeric hEGF are connected downstream of highly efficient transcription and translation signals, and introducing this into E. coli.

The case where m = 2 will be explained with reference to FIG. The hEGF precursor (multimeric hEcF) produced by E. coli is not easily decomposed within the cells. However, on the other hand,
・When trypsin, which is an animal-derived peptide chain hydrolase that strongly cleaves peptides on the C-terminal side of linked basic amino acids (arginine, lysine, and bistidine), acts on it, the multimeric hEGF becomes hEGF. between the C-terminal amino acid arginine and the next lysine,
Alternatively, it is cleaved between the last lysine and the next N-terminal amino acid of hEGF, asparagine, resulting in a large number of monomeric h
EGF molecules are produced. The above-described peptide chain cleavage operation is possible because the above-mentioned locations are the only locations where basic amino acids are continuous in the multimeric hEGF molecule.

m=1. The same applies to the case of 3 or more.

There are two methods for obtaining the structural gene DNA that carries the sequence information of hEGF, which consists of 53 amino acids. (
1) Hybrid DNA from cells that actually produce hEGF
(2) A method of isolating and purifying messenger RNA for hEGF using a pairing method, and biochemically synthesizing DNA from this;
Among the various gene base sequences predicted from the amino acid sequence of In some cases, one method is to select one base sequence that seems to be the most appropriate, taking into consideration whether the structure is difficult to make mistakes, and then synthesize it organically. 5
Since it is much easier to organically synthesize 159 base pairs corresponding to 3 amino acids than to search for them among 3×10 g base pairs (base pairs estimated to exist in human cells), in the present invention, (2 ) method was selected.

Furthermore, in the present invention, an oligolysine gene is interposed between consecutive structural genes of monomeric hEGF, and an SD sequence -ATG, which is a translational control signal of the galactokinase gene derived from Escherichia coli, is provided upstream of the structural gene. DNA fragments identical to the nucleotide sequence of the region were ligated.

hEQ with translation control nucleotide sequence prepared in this way
In order to express the F gene in E. coli, it is necessary to link it to a vector that has a transcription promoter that promotes gene transcription, but the necessary linking nucleotide sequence is also synthesized and can be incorporated into the vector. did.

[Effect]

t&Cp trib and also 3 with cUV promoter
Three types of recombinant plasmids were obtained by integrating all genes including translational control signals and structural genes into different plasmid vectors.

Next, the plasmid was transferred to host E. coli (HBIOI).

JM103. C600) to obtain recombinant E. coli, which was cultured. Multimeric hEGF was detected, recovered, and purified.

Furthermore, the multimeric hEGF was decomposed into monomers using the proteolytic enzyme nitrypsin to obtain monomeric hEGF.

〔Example〕

First, we will describe the DNA sequence design and synthesis of the EGF structural gene and translational control signal region. Figure 2 shows the amino acid sequence of hEGF and the corresponding gene. h
Since the amino acid sequence of EGF was determined by Gregory et al., its structural gene can be predicted from the amino acid/gene codon correspondence table. However, because there are some duplications on the gene codon side, this correspondence is not unambiguous.Therefore, the gene codon frequency table by host (Ikemura, Cell Engineering).

vo! , 2. Na13. pp. 78-89 (1983)
), we selected gene codons that appear frequently in E. coli for each amino acid, and first designed a 159-base structural gene blueprint consisting only of these codons.

Furthermore, a translation initiation codon ATG is provided at the beginning of the hEGF gene, and the upstream region is further divided into the SD sequence-ATG region of the galactokinase gene derived from E. coli (K.
21st Class Matsuke (K, McKennyetaQ) (Gene Amplification and Analysis (Ga)
neA+mplification andAnaly
sis), vol, 2. p. 408 (1981)
).

Next, the nucleotide sequence of a part of the oligolysine linker was set as (AAPI, m)m (where Psgwa is A or G, 1<i<m, and m is a natural number).

A nucleotide sequence (BamHI linker) for linking to an expression vector is connected to both ends of the nucleotide sequence obtained in this way, and finally a translatable multimeric hEG
We designed the gene for F.

Figure 2 shows m=2. Amino acid sequence and DNA sequence of multimeric hEGF when Pl, z=A, Pa, z=G: (1
85+165n) base pair, n is not particularly defined at this stage, but is defined by E. coli cloning (described later).

By the way, long DMA chains exceeding 100 bases cannot be synthesized by a series of DNA chain elongation reactions using the phosphoramid method, which is a known DNA synthesis method. Even if the yield of the elongation reaction for one base is 95%, if it is repeated 30 times, the total yield will be about 21%, making purification difficult. Therefore, in reality, the long double-stranded DNA in Figure 2 is converted into blocks of 22 types of single-stranded oligonucleotides as shown in Figure 3: F1 to F1.
It was divided into 22 parts, and each was synthesized by a DNA chain elongation reaction using the phosphoramid method.

When creating these blocks, care is taken to ensure that when these blocks are combined into one to create the EGF gene, inappropriate interactions between the blocks will not occur, resulting in the formation of a gene with an incorrect structure. , the base sequence of each block was optimized.

FIG. 4 shows the procedure for ligating blocked oligonucleotides using Ta DNA ligase. Since there was a risk that incorrect combinations would occur if 22 fragments were ligated all at once, a conventional method was followed for the 0 reaction, which employed a two-step ligation method.

In this ligation reaction, Bl and B4. B2 and B3 are equimolar, and B2. B3 is Bl.

It is important to add at least the same mole to B4, n, ≧-1
The probability of constructing a multimeric hEGF gene is improved.

Next, according to FIG. 5, the tae of the synthesized hEGF gene
The procedure for molecular cloning into an expression vector with a promoter is shown. First, the β-lactamase gene (Am
p') and the galactokinase gene (G a l K)
as a selectable marker gene, and furthermore, the 35 region of the tryptophan promoter and the P of the lactose promoter.
pDR540, an existing expression plasmid vector that has a tac promoter fused with ribnow sequence.
(a) was cleaved with the restriction enzyme BamHI and then treated with bacterial alkaline phosphatase,
A cleavage cloning vector (b) in which the BamHI end was dephosphorylated was obtained. Furthermore, the aforementioned synthetic multimeric hEGF
Connecting genes (c) and (b) with enzyme T a DNA
Novel recombinant plasmid PGEG ligated by ligase
F2(d) was obtained. Next, E. coli JM103, which was used as a recipient cell using the rubidium-calcium method, was transformed into plasmid (d).
Transformed with This bacterium was cultured on an agar plate medium containing ampicillin, and the resulting colonies (containing the ampicillin resistance gene A m pr , that is, pGEGF2
After culturing and propagating several isolated colonies in a liquid medium, the recombinant plasmid vector (d) was recovered from the bacterial cells (Molecular Cloning).
, Mania Teis et al. 11IA (ed by Mania
Cold Spring Harbor Laboratory
Laboratory), pp. (1982)).

Restriction enzyme B
a m Cut with HI, separate by agarose gel electrophoresis, elute from gel and purify 1M13 phage B a m
The DNA was ligated to the HI site and sequenced by the dideoxy method.

As a result, n=1, 2, 3. The construction of several types of expression vectors containing multimeric hEGF genes was confirmed.

〔Effect of the invention〕

Plasmid vector pGEGF produced according to the present invention
pDR540 (Figure 5(a)), the parent vector of 2, is introduced into Escherichia coli C600pgaQK-pHBIO1, etc., the bacteria are cultured, and galactokinase is produced. It reaches about 50%. This is the transcription efficiency of the galactokinase gene (gaIIK),
This shows that the translation efficiency is extremely high compared to any of the plasmid internal genes and the approximately 2,000 types of genes that the host originally carries.

Plasmid vector pGEGF produced according to the present invention
2 (Fig. 5(d)) has the same transcription promoter and translational control signal as the gaQK of the above pDR540, and hEG
Highly efficient production of F is expected.

In fact, recombinant E. coli C6C60OQ K- was obtained by transformation using a plasmid vector in which n=2.

Culture HBIOI, JM103, etc. to 1 mg/Q
(Culture solution) Production of the above multimeric hEGF was confirmed by radioreceptor assay method (RRA method).

Next, the multimeric hEGF was separated and purified by high performance liquid chromatography using ODS gel as a carrier and an acetonitrile-water eluent.

The thus obtained multimeric hEGF was digested with trypsin obtained from bovine pancreas to obtain monomeric hEGF.

The activity of this sample was measured by RRA method, and the result was approximately 3 mg/
It was confirmed that hEGF corresponding to Q (culture solution) was produced.

[Brief explanation of the drawing]

Figure 1 is a schematic diagram of the process of obtaining monomeric hEGF from multimeric hEGF, Figure 2 is a diagram showing the entire synthesized base sequence, and Figure 3 is a diagram showing the complete base sequence divided into 22 synthetic oligonucleotide blocks. FIG. 4 is a diagram showing the flow of assembly and linkage of synthetic oligonucleotide blocks, and FIG. 5 is an explanatory diagram showing an application example of the present invention. A...Adenine, C...Cytosine, G...Guanine, T Figure 1 Kaya 5 section

Claims (1)

[Claims] 1. DNA sequence: [There is a gene sequence. ] When SEQ is {SEQ+(AAP_i_,_m)_m}_n+SEQ
(P_i_,_m is A or G, 1≦i≦m, m, n
DNA containing a synthetic gene for the production of human epidermal growth factor (wherein is a natural number). 2. In the item described in claim 1, DNA
There is a nucleotide sequence [gene sequence] upstream of the nucleotide sequence. ] DNA. 3. In the item described in claim 2, DNA
Further upstream of the nucleotide sequence is the tac promoter, t
1. A recombinant plasmid, which is characterized in that it has an rp promoter, a lacUV promoter, or some thereof, thereby enabling high-efficiency production of a polypeptide in Escherichia coli cells.