KR920007684B1 - New expression vectors - Google Patents

Abstract

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Description

Novel expression vector and preparation method thereof

1 shows the plasmid obtained above from the EcoR I-HindIII fragment derived from intermediate III.

2a, b and 3 show restriction enzyme sites and specific structures and sequences of plasmids.

The present invention relates to a novel expression vector, and more specifically, β-galacto consisting of 15 to 20 amino acids in addition to the usual transcription and translation control sequences such as promoter, operator, ribosomal binding site, translation start site, transcription termination site, etc. A sequence encoding a portion of the sidase, an operator sequence or a repeat sequence of a portion thereof, an additional ribosome binding site and translational initiation codon of at least one conventional, a sequence encoding a homopolymer oligopeptide consisting of 5 to 10 identical amino acids, and It relates to a novel expression vector comprising ATG codons added in addition to the conventional ones. In addition, the present invention relates to a method for producing such an expression vector.

Any lineage gene carrying any form of information can be introduced into bacterial cells by in vitro genetic recombination methods. Such stable preservation of foreign DNA and expression of information can be performed using a suitable carrier molecule, that is, an expression vector. When a gene encoding a protein is expressed in a bacterial cell, the bacterial enzymes first synthesize mRNA from DNA during transcription, followed by synthesis of the protein from it during translation. Expression begins at a DNA site called a promoter, and RNA polymerase is bound to this site prior to initiation of transcription. If in vitro gene recombination methods can be used to produce a desired level of the desired protein, i.e., if transcription and translation have proceeded to an appropriate level and the resulting mRNA and biosynthetic protein are stable in the host cell, this method is practically practical. It would be very valuable.

Foreign genes (especially eukaryotic genes) are sometimes difficult to express for the following reasons.

Gene product is toxic to host cell

MRNA synthesized from foreign genes is not stable in bacterial cells

Gene products (proteins) decompose very quickly and cannot accumulate.

The first problem of the above can be solved by a promoter that can be regulated and initiated at the moment the user activates when the bacterial cells have reached an appropriate level. In this state, there is no problem if the synthesis of the product continues in the cell and if cell division no longer proceeds, i.e. if the growth conditions are not ideal for the host cell (due to the synthesis process and the toxicity of the product).

In general, mRNA and protein levels can be protected by "masking" the gene in question, ie by fusing a gene from bacteria at the 5'-end of the target product gene. As a peptide of such a protective function, a part of the β-galactosidase enzyme is generally used. Since at least 150 amino acid sequences are required for protection, the ratio of the protective peptide in the fusion protein is high. This relatively decreases the ratio of effective proteins in the fusion protein, and the BrCN cleavage, which is a commonly used separation method, causes a large number of fragments, making it difficult to select the desired ones. Given.

Recently, a method for fusion of short DNA tails encoding homopolymers of identical amino acids at the 5'-end of the gene was developed and reported in E. coli to protect foreign proteins. (Wing L. Sung et al .: Short synthetic oligdeoxyribonucleotide leader sequence enhance accumulation of human proinsulin synthesized in Escherichia coli, Pro. Natl. Acad. Sci. USA.Vol. 83, pp.561-565, 1986). For some amino acids (Thr, Gln, Ser), if only 6 to 8 amino acids are repeated to form a homopolymer moiety, the gene can be expressed very well (6 to 26% of the total protein) and the fusion protein The proportion of effective protein in the liver can be significantly increased. However, the short sequence (3 amino acid length) encoding β-galactosidase reported in the above paper cannot sufficiently protect the level of mRNA, and such a vector cannot be generally used as the vector of the present invention. . According to the report, the authors assumed that only sequences of normal length (coding about 150 amino acids in length) could have such type of stabilizing function based on prior experiments, so short sequences as mentioned above are not important. Did.

Rather, it is surprising that inserting a region encoding the same amino acid between the front of the gene to be expressed and the single-phase β-galactosidase DNA sequence (15 to 20 minanoic acid coding) results in mRNA levels. I found that it can protect me. In other words, these “sandwich” arrays provide sufficient protection for mRNA and protein. Thus, we have concluded that the β-galactosidase coding moiety is important for protecting the level of mRNA and that the homopolymer amino acid tail is important for protecting the level of protein.

Therefore, the present inventors have made efforts to develop a novel expression vector, which guarantees excellent protein yield and is surely regulated and provides sufficient mRNA and protein level stability regardless of the type of gene to be expressed. From the appropriate members of pERVI / 23 (Hungarian Patent Application No. 4111/87), the in vitro DNA recombination method was used to prepare threo-α plasmid pER 23 meeting the above requirements (restriction site of this plasmid). And a functional map is shown in FIG. 2A). The preparation of such plasmids can maintain high levels of mRNA and protein when the inventors use homopolymers consisting of identical amino acids with single-phase sequences encoding β-galactosidase (40 to 60 nucleotides in length). In addition, the introduction of the entire operator portion makes the vector more fully regulated, and the introduction of additional ribosomal binding sites and detoxification sites significantly increases the amount of protein produced by the vector. It comes from one place.

That is, an object of the present invention, in preparing an expression vector by introducing a sequence encoding a homopolymer consisting of identical amino acids, a sequence encoding an operator and an operator sequence encoding 15 to 20 amino acids in β-galactosidase or Some repeating sequences, additional ribosomal binding sites and detoxification codons, sequences encoding homopolymers of 5 to 10 identical amino acids, and additional ATG codons as plasmid vector pERVI / 23 derivatives, promoters, operators, ribosomes thereof It provides a method for preparing an expression vector by introducing between the binding site and the translation start codon moiety and the front of the gene to be expressed.

The characteristics of the expression vector and the gene expression provided by the present invention is as follows.

The vector of the present invention carries a very strong regulatory promoter.

mRNA and protein levels are protected by placing the homopolymer amino acid tail synthesized by the coding sequence of the homopolymer consisting of the same amino acids as the -β-galactosidase coding sequence in front of the gene to be expressed.

The tail length is 40 amino acids, making purification of the product very easy.

Since the ATG codon is inserted before the gene to be expressed, unwanted fusion protein portions can be removed simply by BrCN cleavage of the resulting protein.

A nearly literal second lac operator sequence can be found after the -β-galactosidase portion (found in the leading open frame with the amino acid coding sequence and the β-galactosidase coding portion). This second operator, with the first original operator, was located behind the promoter to form promoter 6/23 (Hungarian Patent Application No. 4111/87) formed with a regulatory sequence of the P2 promoter of the ribosome RNA operon and the E. coli lac operon. Even after strong promoters such as

A second ribosome binding site and detoxification codon can be found after the -β-galactosidase coding portion and before the coding sequence of the homopolymer consisting of the same amino acids. According to the experimental results, it is possible to select a signal that is more efficient and easier to use in the bacterial detoxification device than when both proteins have only one of the first and second decipher signals. This increases the degree of freedom of the system.

The vector of the present invention may be used for expression of any protein gene containing a Cla I linker or any protein gene inserted into the Cla I site of the vector (other restriction sites of the linker may also be used). .

By providing the same amino acid tail, several proteins can be purified by immunological methods.

-Genes that have not been tested so far (human whole insulin, synthetic insulin A and B chains, vascular active intestinal polypeptides) also show very high levels of expression by the vector of the present invention (25 of the total protein content of the cells). To 30%).

The production method of the expression vector according to the present invention consists of the following steps (more detailed description will be described below).

1. Select a peptide coding gene available in cloned form and known to be very unstable in E. coli cells (eg, human whole insulin).

2. Ligation the Cla I linker to the 5'-end of the gene. At this time, the last three nucleotides (ATG) of the linker are positioned immediately before the nucleotide triplet encoding the first amino acid (amino acid methionine encoded by the ATG codon allows BrCN cleavage of the fusion product) The gene starting with the Cla I linker is cloned after the lac regulator sequence (operator and ribosomal binding site) of the appropriate plasmid.

3. Insert a synthetic oligonucleotide containing a portion encoding some amino acid threonine into a specific Cla I restriction site of the plasmid obtained above. When correct insertion is made, the codons of threonines are present in the reading frame with the gene, and the Cla I site is only regenerated at the ends of the oligonucleotides present in close proximity to the gene.

4. From the plasmid obtained above,

-lac regulator part

Oligonucleotides encoding threonine

-Cla I linker and

-gene

DNA fragments containing were digested with appropriate restriction enzymes and cloned into specific PvuII restriction sites of the α-peptide coding portion of these plasmids pERVI / 23 or any derivative thereof. At this time, a specific restriction site (R) was produced at a position corresponding to the PvuII site. In the relevant part of the plasmid thus obtained, DNA parts having the following functions can be found: 6/23 promoter; lac regulator part I; a DNA sequence encoding the first 34 amino acids of an α-peptide; R specific restriction site; lac regulator part II; Coding sequence of a single oligopeptide consisting of threonine; Cla I linker with ATG codons; Genes encoding peptides of stabilizing function.

5. In the next step, the R restriction site was used to linearize the plasmid, and the Bal 31 exonuclease was performed under conditions such that the enzyme could degrade only 10 nucleotides, and the plasmid was then regenerated with DNA ligase. To illusion.

6. After transformation, select clones that express high levels of foreign proteins of the desired size.

7. Using immunological methods (RIA, immunoblots), confirm that the resulting fusion peptides contain the protein to be expressed.

8. Confirm by sequencing the effective nucleotide sequence of the plasmid.

9. Using the Cla I restriction site, replace the gene coding DNA sequence with a polylinker sequence designed to clone any other peptide gene.

The present invention will be described in more detail based on the following examples. However, the following examples do not limit the scope of the invention.

Preparation of Plasmid pER 23 threo vs-α

Three different kinds of plasmids, pSZI 153, pERVI / 23 PLH4, pERVI / 23 (+ ATG), were used as starting materials. Here, the plasmid pSZI 153 is described in the applicant's Hungarian patent application 4363/84, the other two are members of the vector group described in the applicant's Hungarian patent application 4111/87.

The plasmid pSZI 153 was cleaved with Cla I and HindIII restriction enzymes to isolate the DNA fragment encoding human whole insulin, and the nucleotide, triplet derived from the Cla I linker, was encoded on the removed DNA fragment to encode the first amino acid of the protein. Direct connection with codon TTT was allowed.

As a first step, DNA fragments (preinsulin genes) generated by cleavage with Cla I and HindIII restriction enzymes were cloned into the Cla I-HindIII site of the polylinker portion of plasmid pER23 (+ ATG). In the second step, the plasmid cleaved with Cla I, containing codons for the seven threonine amino acids, followed by the Cla I site immediately preceding the preinsulin gene of the plasmid obtained above (hereinafter called intermediate I). Synthetic double stranded oligonucleotides with two sticky ends that can be ligated were inserted. At this time, the oligonucleotide is designed so that when inserted in the correct direction, the threonine codon is located in the reading frame having the whole insulin gene and the Cla I restriction site is reproduced only at the end of the nucleotide located near the gene. (This is necessary when using the specific Cla I restriction site obtained to replace the whole insulin gene with another gene encoding any peptide).

It is noteworthy that, as reported by Sung et al. (See above), there was no expression that could be identified in E. coli clones transformed with plasmids prepared by the method described above (hereinafter referred to as Intermediate II). to be.

In the next step, specific Nsi I restriction enzyme sites of the intermediate II plasmid are transformed with EcoR I restriction sites. At this time, after the Nsi I cleavage, the 3′-protrusion end was cleaved off with a T ₄ polymerase enzyme, and the plasmid was re-cyclized to DNA ligase under conditions in which an excessive amount of EcoR I linker phosphorylated with polynucleotide kinase was present. . The plasmid thus obtained is referred to as intermediate III hereinafter.

Then, the EcoR I linker is inserted again into the plasmid pER23 PLH4 (neutral of the α-peptide coding portion). At this time, the T ₄ polymerase treatment is unnecessary because it has a blunt end produced by the PvuII enzyme.

Subsequently, the EcoR I-HindIII fragment derived from Intermediate III was cloned into the EcoR I-HindIII site of the plasmid obtained above (hereinafter referred to as Intermediate IV). The plasmid thus obtained is referred to as intermediate V below, and the structure of the relevant site is shown in FIG.

In the next step, the intermediate V plasmid was digested with EcoR I restriction enzymes, followed by a series of deletions with Bal 31 exonuclease in both directions from the EcoR I site. At this time, Bal 31 enzyme deletes several dozen nucleotides in both directions, thereby reducing the distance between the α-peptide coding portion and the seven threonine codons.

After re-cyclization with DNA ligase, the resulting mixture of plasmids was transformed into E. coli JM107 cells, and strains producing large amounts of foreign genes of the desired size range were selected (suitable for confirming that the plasmid obtained was correct). Clones were selected and identified by DNA sequencing of the end points of the deletions in the plasmids prepared above, the specific structures and sequences of which are shown in Figures 2a, b and 3. The resulting fusion proteins were human whole insulin Reacted strongly with an antibody having specificity).

As a final step, Cla I-HindIII fragments containing the whole insulin gene were replaced with Cal I-HindIII polylinker fragments of plasmid VI / 23 PLH4 derived from miniplasmid IIVX.

[Drugs and other preparations]

Commonly used experimental drugs were purchased from REANAL, SIGMA and MERCK. γ- ³² P-ATP and α- ³² P-dATR preparations were obtained from the Hungarian Institute of Isotopes (IZINTA, Budapest), and those having a specific activity of 100 to 150 TBq / mM were used.

Restriction enzymes PvuII, EcoR I, HindIII were isolated from the Biological Research Center of the Hungarian Academy of Science according to known methods (Methods in Enznology, Ed; L. Grossmann, V. Moldave, Vol. 65, pp, 89-180). It was. Restriction enzymes Cla I, Nsi I were purchased from New England Biolabs.

BAL 31 exonuclease and cleno-enzyme (E. coli DNA-polymerase I large fragment) were purchased from New England Biolabs, and bacterial alkaline phosphatese (BAP) was purchased from Worthington, Pancrase RNase and lysozyme. Was purchased from REANAL.

T 'nucleotide ligase was prepared according to Murray et al. (Murray, NE, Bruce, SA and Murray, K: Molecular cloning of the DNA ligase gene from bacteriophage T', I. Mol. Biol. 132 (1979), 493 -505).

[Strain]

E. coli K12 JM107 (Yanisch-Perron, C. et al: Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors, Gene 33; (1985), 103-119).

E. coli strains were cultured in a nutrient rich medium containing 10 g of Bakto-Tryptone, 5 g of Bakto yeast extract and 5 g of NaCl per liter. Solid medium was made by adding 15 g of bacto-agar per liter to the liquid medium.

The indicator for evaluating the expression of the N-terminal portion (α-peptide) of β-galactosidase contained the following components per liter.

Strains carrying plasmids containing the β-lactamase coding gene were cultured in a medium containing 100 μg / ml ampicillin.

Isolation of plasmid DNA was performed by culturing bacterial strains carrying the problem plasmids in a medium containing 100 μg / ml ampicillin and amplifying plasmid DNA by adding 170 μg / ml chloramphenicol at 0D _{600 nm} = 0.7-0.8. Laboratory scale plasmid isolation was performed by Clewell and Helinski's method (Clewell, DB and Helinki, DR: Supercoiled circular DNA-protein complex in Escherichia coli: purification and induced conversion to an open circular DNA form.Proc. Natl. Acad Sci. USA 62 (1969), 1159-1166). The resulting clean digest was further purified by ultracentrifugation on Sephacryl S1000 (Pharmacia) chromatography or cesium chloride / ethydium bromide gradient. Analytical scale plasmid separation (from 1.0-1.5 ml bacterium culture) was performed by Birnboin and Doly's potassium acetate method modified by Ish-Horovitz. (Maniatis, T., Fritshc, EF and Smabrook, J., Molecular cloning, Cold Spring Harbor Lab., New York, 1982).

Cleavage of DNA samples using restriction enzymes was performed according to the conditions suggested by New England Biolabs.

Reaction mixture (30-40 μl) containing 0.5-1.0 μg DNA, 66 mM Tris-Hc1 (pH = 7.6), 5 mM MgCl2, 5 mM dithiothreitol, 1 mM ATP and 1 Unit of T₄induced polynucleotide lease Ligation). The ligation mixture was left at 14 ° C. for 2-3 hours (Maniatis, T., Fritsch, E.F. and Sanbrook, J: Molecular cloning, Cold Spring Harbor Lab., New York, 1982).

Dull DNA fragments contain 30-40 μg / ml DNA, 25 mM Tris-HC1 (pH = 7.4), 5 mM MgCl2, 5 mM dithiothreitol, 0.25 mM spermidine, 1 mM ATP, 10 μg / ml BAS (Sigma, typeV) Was ligated in a mixture. After adding 4-6 units of T′-induced polynucleotide lease, the reaction mixture was left at 14 ° C. for 8-12 hours.

Agarose (Sigma, Type I) gel-electrophoresis of DNA samples was performed in a horizontal electrophoresis tank using 0.8-2.0% agarose slab gel according to Helling et al. (1974). Polyacrylamide gel-electrophoresis of DNA samples was carried out in a vertical apparatus using 4 and 8%, 1 mm thick slab gel according to Maniatis et al. (Maniatis, I., Jeffrey, A. and vande Sande, H: Chain length determination of small double and sing-stranded DNA molecules by polyacrylamide gel electrophoresis, Biochemistry 14, (1975) 3787-3794).

DNA fragmentation from agarose gels and polyacrylamide gels was performed using DEAE-paper, according to the method of winberg, G. and Hammarskjold, M. D. (Isolation of DNA from agarose gels using DEAE-paper.Application to restriction site mapping of adenovirus type 16 DNA, Nucl.Acids Res. 8, (1980) 253).

BAL31 exonuclease digestion of DNA fragments was performed at a temperature of 30 ° C. in a reaction mixture containing 100 μg / ml DNA, 600 mM NaCl, 12 mM CaCl 2, 20 mM Tris-HCl (pH = 8.0), 1.0 mM EDTA. Depending on the extent desired to be shortened, 0.4-1.2 units of enzyme were added to the mixture containing 10 μg DNA. Each case of each given DNA fragment was subjected to test reaction with a given enzyme-sample to vary the time left and then gel-electrophoresed to determine the actual shortening rate. The enzymatic reaction was terminated by extracting the reaction mixture with phenol, purified with ethanol and blunted using Klenow polymer raise under conditions suitable for labeling the 3'-terminus of the DNA terminus. (Maniatis, T., Fristsch, Cold Spring Harbor., New York, 1982).

E. coli cells with adequate requirements were prepared according to the CaCl2 method of Mandel and Higa using strain JM107 (Maniatis, T., Fritsch, EF and Sambrook, J: Molecular cloning, Cold Spring Harber Lab, New York, 1982). .

Dephosphorylation reaction at the DNA 5′-end, end-labeling with polynucleotide kinase using γ- ³² P-ATP, and nucleotide sequence analysis were performed according to the method of Maxam, A and Gilbert, W. Sequencing and labeled DNA with basespecific chemical cleavage.Meth.Enzymol. 65, (1980) 499-560).

Insulin-containing fusion proteins have been demonstrated by immunoblot methods such as Towbin (Electrophoretic transfer of proteins from polyacylamide gel to nitrocellurose sheets, procedure and some application, Proc. Notl. Acad. Sci. USA. 76, 43 50 ( 1979)).

Other methods used for the preparation of the vectors according to the invention are described in Maniatis, T, Fritsch, E.F. And the protocol set forth in Sambrook, J.'s Experiment Guide (Molecular cloning, Cold Spring Harbor Lab., New York, 1982).

Abbreviations used in drawings

Pr: Promoter T₁T₂: Transfer Terminator

sp: shine-dalgrino sequence (ribosome binding site)

aa: amino acid

op: lac Operator: Deleted part (by Bal 31 exonuclease)

thr: threonine condon ori: origin of replication

α: α-peptide gene h.p.i. Human whole insulin gene

Apr: β-lactamase gene that provides ampicillin resistance

E: EcoR I restriction enzyme Ba: BamH I restriction enzyme

Pst: Pst I restriction enzyme Bg: Bg1 I restriction enzyme

X: Xba I restriction enzyme H: Hind III restriction enzyme

Claims (8)

In preparing an expression vector by introducing a sequence encoding a single polymer composed of identical amino acids, a sequence encoding an amino acid of 15 to 20 amino acids in β-galactosidase, a repeating sequence of an operator sequence or part thereof, and additional ribosomes The binding site and initiation codon, a sequence encoding a homopolymer of 5 to 10 identical amino acids, and an additional ATG codon into the plasmid vector pERVI / 23 derivative, the promoter, operator, ribosomal binding site and translational initiation codon moieties Method for producing an expression vector, characterized in that introduced between the back and the front of the gene to be expressed. The method of claim 1, wherein pERVI / 23 (+ ATG) is used as the plasmid vector pERVI / 23 derivative. The method of producing an expression vector according to claim 1, wherein a sequence encoding 17 amino acids in β-galactosidase is introduced as a sequence encoding a part of the β-galactosidase. The method of claim 1, wherein a sequence encoding a homopolymer consisting of seven identical amino acids is introduced as a sequence encoding the homopolymer consisting of identical amino acids. The method of manufacturing an expression vector according to claim 1 or 4, wherein a sequence encoding a homopolymer of threonine is introduced as a sequence encoding the homopolymer of identical amino acids. In expression vectors containing sequences encoding homopolymers of identical amino acids, the plasmid pERVI / 23 derivatives are expressed by β- in front of the promoter, operator, ribosomal binding site and detoxification codon moiety and before the gene to be expressed. Sequences encoding 15-20 amino acids in galactosidase and repeating sequences of operator sequences or portions thereof, additional ribosomal binding sites and initiation codons, sequences encoding homopolymers of 5-10 amino acids, and additional ATG codons Expression vector characterized in that it is transformed by containing. The expression vector according to claim 6, wherein the expression vector has a structure as shown below. Where pr is a poromotor, T ₁ T ₂ is a transcription terminator, SD is a shine-dalgrano sequence, ribosomal binding site, op is a lac operator, ▽ is a deletion, thr is a threonine codon, ori Is the origin of replication, α is the α-peptide gene, hpi is the human whole insulin gene, and Ap ^r is the β-lactamase gene that provides ampicillin resistance. Sequences and operator sequences encoding 15-20 amino acids in β-galactosidase between the plasmid pERVI / 23 derivatives after their captive motor, operator, ribosomal binding site and detoxification codon moiety, and before the gene to be expressed Or in an E. coli host strain using a transformed expression vector containing some repeat sequence, additional ribosomal binding site, detoxification codon, a sequence encoding a homopolymer of 5 to 10 amino acids, and an additional ATG codon How to mass produce proteins.