KR840000949B1 - Method of making aselected protein - Google Patents

Abstract

A plasmid for a periplasmic or extracellular bacterial protein was cleaved; a double-stranded DNA sequence coding for a selected protein or protion thereof from a eukaryotic cell, such as insulin, was inserted in that cleaved gene by recombinant DNA technique and used to transform a bacterium, and the excreted selected protein was collected. Thus, double stranded cDNA prepd. from RNA contg. preproinsulin in RNA isolated from a B-cell tumor was inserted into an Escherichia coli penicillinase gene.

Description

Method for producing recombinant protein

1, 2, and 3 are graphs showing the complete amino acid chain of the E.Coli penicillinase gene performed on plasmid pBR 322 but not the corresponding amino acid chain of the protein.

By preparing a specific protein from the bacterium of the invention, and then discharging it from the bacterial cell, and further by inserting the DNA representing the desired protein by the plasmid or phage gene recombination technique of the perplasmic or cellular protein, and into the recombined DNA A method of mutating a bacterial host and culturing the mutated host to release the desired protein.

The protein thus produced can be collected by conventional methods in a periplasmic space or incubator depending on the type of media protein gene.

Methods of incorporating DNA expressing specific proteins into genes for intracellular proteins are well known (Schler et al., 196, p177-180 (1077). However, proteins produced by these methods are not compatible with other intracellular proteins). There has been a problem of collecting proteins in purified form because they are mixed and thus degraded by intracellular enzymes.

These problems can be overcome by the present invention, a method of inserting DNA expressing a desired protein into a bacterial gene. That is, the bacterial gene for the cell or periplasmic protein is deleted, non-bacterial DNA (indicating the desired protein) is inserted by the recombination technique, and the recombined gene is used to mutate the bacterial host. It can be overcome by culturing the mutated host to release the desired protein.

As an example, it has a leader chain of hydrophobic amino acids in an amino terminal group and uses a gene for a medium protein that flows out through the cell membrane, so that the hydrophobic leader chain cracks the selective protein according to the selection of the medium protein. Or it can be recovered from the medium in which the bacteria grow.

The method described above prevents contamination from other proteins in bacteria and greatly increases stability because they are not in contact with bacterial intracellular enzymes that contaminate the desired protein.

Bacterial genes for media proteins that can be used in the present invention include penicillin resistant or penicillinase gene, chromamphenicol gene, tetracycline resistance gene and alkaline phosphatase gene and ribonuclease gene. .

Genes or DNA fragments that code for various proteins can be inserted into bacterial media protein genes by the method of the present invention. Such proteins include various nonbacterial proteins such as eukaryotic cell proteins and viral proteins. Of particular importance are eukaryotic proteins such as insulin, growth hormone, interferon and other pharmaceutically active proteins. It is synthesized into these individual genes, such as precursor proteins with amino acid sequences at the amino terminus. The hydrophobic leader sequence is not identical to the bacterial protein that is cabbed through the bacterial membrane. Therefore, due to the fact that higher cell preinsulin or other pre-proteins have hydrophobic leader chains, there is no expectation that they can be matured in bacterial cells themselves even if they can be synthesized intracellularly. . In addition to the synthesis and excretion in the bacterial cells of the mature protein of known eukaryotic cells, which are now in practical use, the method of the present invention allows the synthesis and excretion of bacterial products of other cellular products of commercial interest.]

It also encompasses special crystalline proteins such as virus antigen proteins or other molten proteins consisting of media proteins with capsular proteins as defined above. The above-mentioned molten protein is useful for preparing a vaccine due to antigenicity such as being able to cause an antibody reaction against viruses. The vaccine is rarely safe because it does not contain any active or inert virus. Moreover, the vaccine for viruses which cannot be cultured and grown by this method can also be prepared.

The following examples are intended to illustrate the nature of the invention without limiting its scope.

EXAMPLE

The gene operating in the small plasmid pBR 322 was used as the medium protein E. coli Escherichia coli] penicillinase. Restriction enzyme diagrams of the genes are shown in the figure. The plasmid vector is described in Bolivar Seed Genes 295-113 (1977).

As a host bacterium E. Coli × 1776; See Recombinant Injection of Curtis and Jess; The impact on science and society, the Ten Smile International Symposium Publications Beer & Versette 45-56 (1977).

Host-beta binding was confirmed by the National Institutes of Health (NIH) on July 7, 1977.

The plasmid carries Pst (ProVideucia stuartii endonuclease) restriction of the penicillinase gene corresponding to the positions of amino acids 181 and 182 as shown in the figure.

The double helix cDNA was RNA-containing prepro insulin in x-ray induced transplanted rat B-cell tumors (PPI mRNA) isolated from hick et al., Proc, Natl, Acad, Sci, US [pNAS] 74,628 (1977). Synthesized from

A batch of 20 grams of each frozen tumor fragment was dispensed with Mg ⁺¹ precipitate (palm, biochemistry 13,3603-3615 (1974) supernatant, extracted from mortar, pestle and phenol and chloroform) with cytoplasmic RNA purified. Same as above (F. a.) Except that it was purified by aligo-dT-cellulose chromatography (PNAS of Avivze, 69 p1408-1412 (19721) and a special (dpT) 8dpGp Fourmer (co-research) was used as reverse transcription) Stradis seed cells 7 279-288 (1976) Directly used as template for double-helix cDNA synthesis, RNA and primer concentrations of 7 mg / μl and 1 mg / μl, respectively.

All four α-32p-dNTPs are 1.25 mM (final specific activity of 0.85 ci / mmol).

Reverse transcription was 2% of the injected RNA and 25% of which was recovered in the double helix DNA product.

Double helix DNA was inserted into the Pst locus of plasmid pBR 322 by the following method. pBR 322 (5.0 μg) was linearized with Pst and approximately 15 dG residues were ended at 15 ° C. in the presence of 1 mMCo ^{+ 2} (Lycolyticase. nucleic acid Res. 3, 101-116 (1976) and 100 μg / ml autoclave). Transferase was added per 3 'end.

The dC residue was added to 2.0 μg double helix DNA using the same method. The reaction mixture was extracted with phenol and precipitated with ethanol. dC-labeled double helix DNA was electrophoresed on 6% polyacrylamide gels in nature. In the following radiomagnetic method, molecules of 300-600 base pairs (0.5 μg) in size are not captured in the gel (Fstratidis's Multiple Molecular Biology Method, 8, 1-124 (1976). Precipitated, concentrated, redissolved in 10 mM Tris (pH 8), mixed with 5 μg dG indicated pBR322 and dialyzed as 0.1 M NaCl 10 mM EDTA 10 mM Tris (pH 8).

The mixture is heated (4 ml) at 56 ° for 2 hours and then bonded at 42 ° for 2 hours. Hybrid DNA was used for the E.Coli 1776 modification. Inserts can be deleted using oligo dC-dG associations, resulting in Pst cuts.

Modification of the E. Coil × 1776 (Ek-2 host with pBR 322, Ek-2 vector) of a p3 physiologic facility consistent with the N, I, H guidance of the recombination DNA study published July 7, 1976 by the Federal Patent Office It was carried out in a bioload safety cabinet.

E. Coil × 1776 was then modified by a slightly modified transfection method. E. Coli × 1776 is L broth supplemented with 10 μg / ml diamino-pimelic acid and thyminin (Sigma) against 40 μg / ml of 0.5A 590 (10 g Triptone, 5 g Yeast Extract, 5 g NaCl (Difco)). Grew in. 200 ml of cells were precipitated at 3999 rpm and 70 mM MnCl, 40 mM NaOAc. Stirred and resuspended in a 1/10 volume of a buffer buffer containing pH5.6,30 mM CaU ₂ and maintained for 20 minutes on ice. Cells were resuspended in 1/30 volume of the buffer solution.

Conjugated DNA (2 ml) was added to the cells. Purified water (0.3 ml) of this mixture was added to a sterilization test and incubated for 60 minutes on ice. The cells were then placed at 37 ° C. for 2 minutes. The culture was added to each test tube (7 ml) and incubated at 37 ° for 15 minutes. 200 μl of cells are spread on a cellulose filter (Milli pore) with a volmoni on a top plate containing 15 μg / ml tetracycline (filter is boiled to remove impurities before use). Plates are incubated at 37 ° C. for 48 hours. A duplicate of the filter was made. The nitro cellulose filter containing the variant was removed from the agar and placed on sterile wort only on filter paper. The new sterilization filter was placed on top of the cell-containing filter and pressurized with a sterile velvet cloth and a double block. Sterile needles were used for the filters. The second filter was placed on a fresh top plate and incubated at 37 ° C. for 48 hours. Cells on the first filter were treated with the Grunsustein-Hognis method (PNAS 72 3961-) using a 80-nucleotide-length probe produced by high specific activity of Hal HI enzymatically copied from murine oligo-dT bound RNA. 3965 (19751).

Positive cells were refiltered by the HART method (Peterson's majority PNAS 74, 4370-4374 (1977) as follows.

Plasmid DNA (about 3 μg) was enzymatically digested with Pst, precipitated with ethanol and directly dissolved in 20 μl of deionized formamide. Each stream was heated at 95 ° for 1 minute and then placed on ice. After addition of 1.5 μg oligo (dT) -cellulose bound RNA, the mixture was added to 10 mM PIPES and 0.4M NaCl at pH 6.4, and the mixture was incubated at 50 ° for 2 hours. It was diluted with 75 μl water, precipitated in the presence of 10 μg Whit-Germ + tRNA, washed with 70% ethanol, dissolved in H ₂ O and a Wheat-Germ cell glass modified mixture (Robert's many PNAS 70 2330-2334 (1973) Was added.

After 3 h at 23 ° C., 2 μl purified water was removed for trichloro acetic acid precipitation; The remainder of the reaction mixture is treated with ribonucleases; Diluted with immunoassay buffer and analyzed for synthesis of immunoreactive prepro-insugen with dual antibody immunoprecipitation (Rometic's multiple nucleic acid Res 3 381-391 (1976). The washed immune precipitate was analyzed with 1 ml of NCS (Emersham). ) And measured in 10 μl of Omnifloor (New England Nucleus) by liquid scintillation.

One colony was confirmed by HART screening.

The ablation insert was chained by the Mexam and Gilbert method (PNAS 74 560-564 (1977) to show that it was linked to preproinsulin 1. The insert was labeled by a nick modification with DNA polymerase 1, Gunstein-Hogny It was used in a 200-transformant sieve with bovine experiments.

48 clones were identified, including murine preproinsulin DNA insertion.

48 clones of the modified E.Coli × 1776 in situ were subjected to radioimmunoassay to determine if cron produced a fusion polypeptide chain of the insulin antigen, one end was an insulin antigen and the other was a penicillinase. (Bacterial medium protein) Determine if it is antigen. As the fused polypeptide chain appears, it shows that the cron contains a gene that is a fusion product of the bacterial gene for penicillinase with the eukaryotic cell gene for insulin. If the molten polypeptide is actually found, the following method is used.

The present invention has the advantage that the fused protein has two antigenic termini, each terminus an antibody terminus linked to a particular antibody. Specific antibodies are placed on elastic plates and the antigenic proteins from the conjugated bacterial cells are placed in contact with the plates, after which the disks are cleaned and exposed to radioantibodies.

The protein molecule is linked to the antibody immobilized in the plasma with one antigenic variant and bound again to the radioantibody with the second variant. If the anti-penicillinase is on the disc and the anti-insulin is connected, the only point of residual radioactivity after the “santwich” is washed will indicate the molten protein. Detailed description of the invention is as follows;

Each of 8.25 cm diameter rolls of clear polyvinyl (Pr) with a thickness of 8 mm were pressed between soft papers (Dorame, New York). In glass petripan, each disk was placed on the side of a solution containing 10 ml of 0.2 M NaHCO ₃ at pH 9.2 containing 60 μg / ml IgG. After holding at room temperature for 2 minutes or more, the disc was removed and washed with 10 ml of cold water buffer (WB), which was composed of phosphate buffered saline, 0.5% guinea pig serum, 21% antiserum albumin, and 0.3 mg / ml streptomycin sulfate. . Each disc was used immediately after cleaning.

Antigens were dissociated from bacterial cells as colonies were transferred to 1.5% agarose containing 0.5 mg lysozyme / ml, 30 mM Tris pH8, and 10 mM EDTA. The IgG-coated side of the PV disks were placed under agarose and bacterial colonies and left for 60 minutes at 4 °.

Each disc was removed and washed three times with 10 ml of cold WB. This step completes the immunoabsorbability of the antigen in the solid antibody layer.

The reaction between the 125 I-labeled antibody and the antigen attached to the disk was carried out at 5 × 10 6 cpm (γ release). Let go. The mesh acts as a spacer. The disk treated in the initial stage is placed in contact with the mesh and the solution and incubated at 4 ° C.

Each disc was washed with 10 ml of cold WB, twice with water and dried at room temperature. The molten protein then contacts the top of the IgG and the radiolabeled layer.

These proteins were detected by conventional autoradiography using a DuPont Kronex Lightning Prussing Screen recorded in the Examples of Kodak Screenfilm or Kodak X-OMAT R Film and Lasker et al., FEBS, Lett, 82 314-316 (1977). It was.

This process is required for the antiinsulin and anti-penicillinase IgG moieties. Antiinsulin antisera are usable products obtained from Guinea. Rabbit anti-penicillinase serum is generated by injecting (1 mg) penicillinase (a complete Freud's adjuvant (Dipco)) into New Zealand white rabbits. (The booster leakage was performed with an incomplete Furoid supplement (Diffco).) 2-3 weeks after the initial injection, rabbits bleed 1 week later.

IgG portions were prepared from angular serum by DEAE-cellulose (Wartman DE-52) chromatography on 0.025 M potassium phosphate pH7.3 1% glycerol followed by ammonium sulfate precipitation.

항원 혈청부피의 0.025M 인산포테슘 pH7.3 0.1M NaCl 1% 글리세롤에 재현탁하고 동완충용액에 대하여 투석하였다. 투석후에 잔사를 원심분리하여 제거하였다. IgG 부분을 70°에서 부분표본에 저장하였다.The flow rate was allowed to pass and the part containing the material was filled and the protein precipitated by addition of 40% ammonium sulfate. The resulting pellets

The serum volume of the antigen was resuspended in 0.025M potassium phosphate pH7.3 0.1M NaCl 1% glycerol and dialyzed against the same buffer solution. After dialysis the residue was removed by centrifugation. IgG portions were stored in aliquots at 70 °.

Each IgG portion was radio-iotized according to the conventional method of Scintilla et al. Biochemistry, 91,943-46 (1964).

The 25 μl reaction mixture contains 0.5 M potassium phosphate, pH 5.5, 2 mci medium free NaI 125 I, 150 μg IgG and 2 μg Chloramine T. After 3 minutes at room temperature 8 μg of sodium metabisulfite dissolved in 25 μl PBS was added and 200 μl PBS containing 2% normal guinea pig cerium.

125 I-label IgG was purified by chromatography on Sephadex G-50 column equilibrated with PBS containing 2% normal guinea pig cerium. The 125I-IgG eluate was whitened to 5 ml with 10% normal guinea pig serum, filtered through a sterile multi-caliber VC filter (0.1 μm diameter), divided into aliquots and stored at 70 ° C.

Special activities are 1.5 x 107 cpm / μg.

The detection method detects a crop of synthetic molten protein × 1776 showing penicillinase and insulin antigenic factors. The protein is absorbed from the foreign material and is consistent with insulin in radioimmunoassay. In the DNA chain, the protein is lysed between penicillinase and proinsulin, and the two proteins are linked to glycine 6 between amino acid 182 of penicillinase (alamine) and glutamine amino acid 4 of proinsulin. Thus, high cell hormones are synthesized as bacteria in antigen-active form.

The DNA chain of the preferred eukaryotic cell protein can be inserted into the tag cleavage at the Hind II cleavage or at the amino acid 45 position relative to amino acids 101 and 102 of the protein to which the pBR 322 plasmid is linked.

In all cases, if the eukaryotic DNA is labeled randomly or arranged by other means, the DNA may be inserted into the gene at a special store by inserting it into a new restriction cut that is convenient for grafting the melt of the media protein. It can be modified by recombination DNA techniques or by mutations. For example, the RI cleavage on plasmid pBR 322 can be removed by mutation and the RI chain can be inserted by suture into the penicillinase gene. Although the above is inert to genes it will not interfere with the use of DNA regions to synthesize media proteins.

Penicillinase DNA manipulation between the code for amino acid 23 and the coat of amino acid 45 at the tag cleavage at the end of the hydrophobic leader can be eliminated as it is nibbled behind the DNA by a combination of suitable enzymes.

One of these mixtures is lambda exonuclease, which degrades the DNA flaky at the 5 'end and is accompanied by the enzyme Sl, which again eliminates a single helix overhang. Another mixture is a T4DNA polymerase that degrades at the 3 'end of the DNA helix with Sl, which in turn eliminates a single helix overhang. By regulating enzymatic digestion, the plasmid DNA molecule can be shortened to fragments protruding from the Rl cleavage to the junction of amino acid 23 or to the point of the hydrophobic leader chain and the fragment can be dissolved in the insulin chain-containing analogous fragment.

The two fragments are fused together by butt and ligation by T4DNA conjugatease and inserted into the plasmid.

This melting produces the degraded species of the media protein, where the media protein is the code for the E. Coli hydrophobic leader sequence and the eukaryotic cell provides the rest of the structural text. Although this structuring is done correctly, the end points can be traced to close-up clots around the medium of bacteria that have been transformed into molten moieties that track antigenic activity by radioimmunoassays as described above. This structure is achieved in a random state.

The method of the present invention is not limited to the E. coli penicillinase gene but can be applied to the gene for the extracted protein on a multicopy plasmid or phage. In addition to insulin, it can also be used to track the expression of molten protein in DNA of viruses or eukaryotic cells that induce coding regions that direct the coding for antigenic determinants in viruses or eukaryotic cells.

Thus, if fragments of animal virus DNA are inserted at the Pst or Hind II sites of the penicillinase gene, some bacteria will synthesize soluble proteins using radioimmunoassays using antibodies to bacterial antigens.

In other words, the molten protein can in turn be purified and used to stimulate antibody responses in animals or humans, such as vaccinating against viruses or producing antibodies directed to specific locations against viruses.

Assuming that only the aggregated state of the molten protein can be used in the vaccine, it is active as a determinant that can assist the immune response in vaccination.

Specific media proteins that can be used are those that can be used for melting of bacterial proteins with sequences that provide useful crystals in their own bacterial proteins or in media proteins.

Claims (1)

Deletes the bacterial gene of an extracellular or extracellular medium, and a non-bacterial DNA portion indicating the coding for the non-bacterial protein or portion thereof to be selected at the deleted position. To insert a hybrid gene to produce a hybrid gene, and to mutate the bacterial host with the hybrid gene, thereby culturing the host to secrete the protein of interest, or a portion or portion of the molten protein, to be selected from the host. A method for producing a desired protein or part thereof by inserting DNA encoding the protein or part thereof into a bacterial gene.

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