WO2023193837A1 - Expression vector for production of recombinant proteins in prokaryotic host cells - Google Patents
Expression vector for production of recombinant proteins in prokaryotic host cells Download PDFInfo
- Publication number
- WO2023193837A1 WO2023193837A1 PCT/CZ2022/050100 CZ2022050100W WO2023193837A1 WO 2023193837 A1 WO2023193837 A1 WO 2023193837A1 CZ 2022050100 W CZ2022050100 W CZ 2022050100W WO 2023193837 A1 WO2023193837 A1 WO 2023193837A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- plasmid
- sequence
- protein
- seq
- gene
- Prior art date
Links
- 239000013604 expression vector Substances 0.000 title claims abstract description 24
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 title claims abstract description 21
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 title claims abstract description 21
- 238000004519 manufacturing process Methods 0.000 title abstract description 26
- 239000013612 plasmid Substances 0.000 claims abstract description 138
- 108090000623 proteins and genes Proteins 0.000 claims description 125
- 102000004169 proteins and genes Human genes 0.000 claims description 65
- 229960000318 kanamycin Drugs 0.000 claims description 22
- 229930027917 kanamycin Natural products 0.000 claims description 21
- 229930182823 kanamycin A Natural products 0.000 claims description 21
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 claims description 20
- 241000588724 Escherichia coli Species 0.000 claims description 19
- 108090000988 Lysostaphin Proteins 0.000 claims description 16
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 16
- 238000010367 cloning Methods 0.000 claims description 13
- 238000000034 method Methods 0.000 claims description 12
- 108091026890 Coding region Proteins 0.000 claims description 11
- 108090000848 Ubiquitin Proteins 0.000 claims description 11
- 102000044159 Ubiquitin Human genes 0.000 claims description 11
- 101800000120 Host translation inhibitor nsp1 Proteins 0.000 claims description 10
- 101800000517 Leader protein Proteins 0.000 claims description 10
- 101800000512 Non-structural protein 1 Proteins 0.000 claims description 10
- 230000003115 biocidal effect Effects 0.000 claims description 10
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 claims description 9
- 239000013613 expression plasmid Substances 0.000 claims description 7
- 239000002773 nucleotide Substances 0.000 claims description 7
- 125000003729 nucleotide group Chemical group 0.000 claims description 7
- 238000004113 cell culture Methods 0.000 claims description 6
- 238000012258 culturing Methods 0.000 claims description 2
- 238000003306 harvesting Methods 0.000 claims description 2
- 244000005700 microbiome Species 0.000 claims description 2
- 230000001131 transforming effect Effects 0.000 claims description 2
- 239000002028 Biomass Substances 0.000 abstract description 6
- 235000018102 proteins Nutrition 0.000 description 56
- 210000004027 cell Anatomy 0.000 description 43
- 108020004414 DNA Proteins 0.000 description 37
- 230000010076 replication Effects 0.000 description 22
- 230000014509 gene expression Effects 0.000 description 19
- 239000013598 vector Substances 0.000 description 16
- 108091008146 restriction endonucleases Proteins 0.000 description 14
- 239000003242 anti bacterial agent Substances 0.000 description 12
- 229940088710 antibiotic agent Drugs 0.000 description 12
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 12
- 239000002609 medium Substances 0.000 description 12
- 230000014616 translation Effects 0.000 description 11
- 238000013019 agitation Methods 0.000 description 10
- 239000000872 buffer Substances 0.000 description 9
- 239000000203 mixture Substances 0.000 description 9
- 229920001817 Agar Polymers 0.000 description 8
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 8
- 239000008272 agar Substances 0.000 description 8
- 239000008103 glucose Substances 0.000 description 8
- 239000007788 liquid Substances 0.000 description 8
- 230000003287 optical effect Effects 0.000 description 8
- 230000009466 transformation Effects 0.000 description 8
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 7
- 125000003275 alpha amino acid group Chemical group 0.000 description 7
- 238000001962 electrophoresis Methods 0.000 description 7
- 239000008188 pellet Substances 0.000 description 7
- 239000013615 primer Substances 0.000 description 7
- 238000000746 purification Methods 0.000 description 7
- 239000012634 fragment Substances 0.000 description 6
- 238000010348 incorporation Methods 0.000 description 6
- 238000002955 isolation Methods 0.000 description 6
- 108700010070 Codon Usage Proteins 0.000 description 5
- 238000007399 DNA isolation Methods 0.000 description 5
- 239000006142 Luria-Bertani Agar Substances 0.000 description 5
- 238000003776 cleavage reaction Methods 0.000 description 5
- 238000002474 experimental method Methods 0.000 description 5
- 230000000977 initiatory effect Effects 0.000 description 5
- 230000007017 scission Effects 0.000 description 5
- 108020004705 Codon Proteins 0.000 description 4
- 102000012410 DNA Ligases Human genes 0.000 description 4
- 108010061982 DNA Ligases Proteins 0.000 description 4
- 238000001712 DNA sequencing Methods 0.000 description 4
- 241001198387 Escherichia coli BL21(DE3) Species 0.000 description 4
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 4
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 4
- 235000011130 ammonium sulphate Nutrition 0.000 description 4
- 239000001166 ammonium sulphate Substances 0.000 description 4
- 229940041514 candida albicans extract Drugs 0.000 description 4
- 239000007795 chemical reaction product Substances 0.000 description 4
- 238000002512 chemotherapy Methods 0.000 description 4
- 238000010790 dilution Methods 0.000 description 4
- 239000012895 dilution Substances 0.000 description 4
- 230000006870 function Effects 0.000 description 4
- 239000000499 gel Substances 0.000 description 4
- 239000002054 inoculum Substances 0.000 description 4
- 239000008101 lactose Substances 0.000 description 4
- 238000010899 nucleation Methods 0.000 description 4
- 239000008363 phosphate buffer Substances 0.000 description 4
- 239000013600 plasmid vector Substances 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 230000035939 shock Effects 0.000 description 4
- 230000000087 stabilizing effect Effects 0.000 description 4
- 238000013518 transcription Methods 0.000 description 4
- 230000035897 transcription Effects 0.000 description 4
- 239000012137 tryptone Substances 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- 239000012138 yeast extract Substances 0.000 description 4
- SGHZXLIDFTYFHQ-UHFFFAOYSA-L Brilliant Blue Chemical compound [Na+].[Na+].C=1C=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C(=CC=CC=2)S([O-])(=O)=O)C=CC=1N(CC)CC1=CC=CC(S([O-])(=O)=O)=C1 SGHZXLIDFTYFHQ-UHFFFAOYSA-L 0.000 description 3
- 102100021935 C-C motif chemokine 26 Human genes 0.000 description 3
- 101000897493 Homo sapiens C-C motif chemokine 26 Proteins 0.000 description 3
- 108010076818 TEV protease Proteins 0.000 description 3
- 230000003321 amplification Effects 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 230000001580 bacterial effect Effects 0.000 description 3
- 230000033228 biological regulation Effects 0.000 description 3
- 239000006285 cell suspension Substances 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 230000000295 complement effect Effects 0.000 description 3
- 238000009826 distribution Methods 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 238000001976 enzyme digestion Methods 0.000 description 3
- 230000002068 genetic effect Effects 0.000 description 3
- 238000002844 melting Methods 0.000 description 3
- 230000008018 melting Effects 0.000 description 3
- 238000003199 nucleic acid amplification method Methods 0.000 description 3
- 108090000765 processed proteins & peptides Proteins 0.000 description 3
- 238000003753 real-time PCR Methods 0.000 description 3
- 230000009467 reduction Effects 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 3
- 238000010186 staining Methods 0.000 description 3
- 238000003756 stirring Methods 0.000 description 3
- 239000012130 whole-cell lysate Substances 0.000 description 3
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 2
- 230000004543 DNA replication Effects 0.000 description 2
- 239000006137 Luria-Bertani broth Substances 0.000 description 2
- 241000204031 Mycoplasma Species 0.000 description 2
- 206010035664 Pneumonia Diseases 0.000 description 2
- 229920001213 Polysorbate 20 Polymers 0.000 description 2
- 108091030071 RNAI Proteins 0.000 description 2
- 101710088839 Replication initiation protein Proteins 0.000 description 2
- 238000005273 aeration Methods 0.000 description 2
- 238000001261 affinity purification Methods 0.000 description 2
- 229960000723 ampicillin Drugs 0.000 description 2
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 230000027455 binding Effects 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000000855 fermentation Methods 0.000 description 2
- 230000004151 fermentation Effects 0.000 description 2
- 102000037865 fusion proteins Human genes 0.000 description 2
- 108020001507 fusion proteins Proteins 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- 238000009630 liquid culture Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 230000000813 microbial effect Effects 0.000 description 2
- 230000035772 mutation Effects 0.000 description 2
- 230000002018 overexpression Effects 0.000 description 2
- 201000006509 pleuropneumonia Diseases 0.000 description 2
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 2
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 2
- 238000001742 protein purification Methods 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- -1 100 mM Tris-HCl pH 8 Chemical compound 0.000 description 1
- 101150015361 5.9 gene Proteins 0.000 description 1
- 101710154825 Aminoglycoside 3'-phosphotransferase Proteins 0.000 description 1
- 108020004217 Aminoglycoside phosphotransferase Proteins 0.000 description 1
- 108020005544 Antisense RNA Proteins 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 108091032955 Bacterial small RNA Proteins 0.000 description 1
- 101800001415 Bri23 peptide Proteins 0.000 description 1
- 101800000655 C-terminal peptide Proteins 0.000 description 1
- 102400000107 C-terminal peptide Human genes 0.000 description 1
- 241000186246 Corynebacterium renale Species 0.000 description 1
- 102000053602 DNA Human genes 0.000 description 1
- 108010017826 DNA Polymerase I Proteins 0.000 description 1
- 102000004594 DNA Polymerase I Human genes 0.000 description 1
- 102000003844 DNA helicases Human genes 0.000 description 1
- 108090000133 DNA helicases Proteins 0.000 description 1
- 102000004163 DNA-directed RNA polymerases Human genes 0.000 description 1
- 108090000626 DNA-directed RNA polymerases Proteins 0.000 description 1
- 238000012286 ELISA Assay Methods 0.000 description 1
- 101000925662 Enterobacteria phage PRD1 Endolysin Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 241000193385 Geobacillus stearothermophilus Species 0.000 description 1
- 101000636168 Grapevine leafroll-associated virus 3 (isolate United States/NY1) Movement protein p5 Proteins 0.000 description 1
- 108010025815 Kanamycin Kinase Proteins 0.000 description 1
- 108090000131 Metalloendopeptidases Proteins 0.000 description 1
- 102000003843 Metalloendopeptidases Human genes 0.000 description 1
- MSFSPUZXLOGKHJ-UHFFFAOYSA-N Muraminsaeure Natural products OC(=O)C(C)OC1C(N)C(O)OC(CO)C1O MSFSPUZXLOGKHJ-UHFFFAOYSA-N 0.000 description 1
- 108700026244 Open Reading Frames Proteins 0.000 description 1
- 238000002944 PCR assay Methods 0.000 description 1
- 108010013639 Peptidoglycan Proteins 0.000 description 1
- 108091000080 Phosphotransferase Proteins 0.000 description 1
- 108010076504 Protein Sorting Signals Proteins 0.000 description 1
- 239000013616 RNA primer Substances 0.000 description 1
- 241000191967 Staphylococcus aureus Species 0.000 description 1
- 241000191978 Staphylococcus simulans Species 0.000 description 1
- 241000282898 Sus scrofa Species 0.000 description 1
- 108010008038 Synthetic Vaccines Proteins 0.000 description 1
- 108010006785 Taq Polymerase Proteins 0.000 description 1
- 241000589596 Thermus Species 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 230000003044 adaptive effect Effects 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 235000001014 amino acid Nutrition 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 239000002647 aminoglycoside antibiotic agent Substances 0.000 description 1
- 102000006646 aminoglycoside phosphotransferase Human genes 0.000 description 1
- 230000000845 anti-microbial effect Effects 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 230000001420 bacteriolytic effect Effects 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 150000001768 cations Chemical class 0.000 description 1
- 238000011072 cell harvest Methods 0.000 description 1
- 230000033077 cellular process Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000013522 chelant Substances 0.000 description 1
- 210000000349 chromosome Anatomy 0.000 description 1
- 230000001332 colony forming effect Effects 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 239000003184 complementary RNA Substances 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 231100000433 cytotoxic Toxicity 0.000 description 1
- 230000001472 cytotoxic effect Effects 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 239000000539 dimer Substances 0.000 description 1
- 230000003828 downregulation Effects 0.000 description 1
- 238000011143 downstream manufacturing Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000001516 effect on protein Effects 0.000 description 1
- 239000012149 elution buffer Substances 0.000 description 1
- 239000002158 endotoxin Substances 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 102000034356 gene-regulatory proteins Human genes 0.000 description 1
- 108091006104 gene-regulatory proteins Proteins 0.000 description 1
- 238000010362 genome editing Methods 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 230000002949 hemolytic effect Effects 0.000 description 1
- 235000014304 histidine Nutrition 0.000 description 1
- 150000002411 histidines Chemical class 0.000 description 1
- 238000000265 homogenisation Methods 0.000 description 1
- 238000009396 hybridization Methods 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 238000012151 immunohistochemical method Methods 0.000 description 1
- 238000009830 intercalation Methods 0.000 description 1
- 230000002687 intercalation Effects 0.000 description 1
- 238000011901 isothermal amplification Methods 0.000 description 1
- 238000011005 laboratory method Methods 0.000 description 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- 231100000518 lethal Toxicity 0.000 description 1
- 230000001665 lethal effect Effects 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 230000002906 microbiologic effect Effects 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- SLCVBVWXLSEKPL-UHFFFAOYSA-N neopentyl glycol Chemical compound OCC(C)(C)CO SLCVBVWXLSEKPL-UHFFFAOYSA-N 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 238000000424 optical density measurement Methods 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 230000036284 oxygen consumption Effects 0.000 description 1
- MXHCPCSDRGLRER-UHFFFAOYSA-N pentaglycine Chemical compound NCC(=O)NCC(=O)NCC(=O)NCC(=O)NCC(O)=O MXHCPCSDRGLRER-UHFFFAOYSA-N 0.000 description 1
- 230000026731 phosphorylation Effects 0.000 description 1
- 238000006366 phosphorylation reaction Methods 0.000 description 1
- 102000020233 phosphotransferase Human genes 0.000 description 1
- 229920002704 polyhistidine Polymers 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 210000001236 prokaryotic cell Anatomy 0.000 description 1
- 230000000644 propagated effect Effects 0.000 description 1
- 230000007026 protein scission Effects 0.000 description 1
- 230000004850 protein–protein interaction Effects 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 229940124551 recombinant vaccine Drugs 0.000 description 1
- 101150034434 repE gene Proteins 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 230000009870 specific binding Effects 0.000 description 1
- 230000003019 stabilising effect Effects 0.000 description 1
- 230000010473 stable expression Effects 0.000 description 1
- 229940037648 staphylococcus simulans Drugs 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 239000003053 toxin Substances 0.000 description 1
- 231100000765 toxin Toxicity 0.000 description 1
- 108700012359 toxins Proteins 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- 230000003827 upregulation Effects 0.000 description 1
- 230000001018 virulence Effects 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
- C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/95—Fusion polypeptide containing a motif/fusion for degradation (ubiquitin fusions, PEST sequence)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/185—Escherichia
- C12R2001/19—Escherichia coli
Definitions
- the present invention provides a novel expression vector particularly suitable for the production of recombinant proteins in prokaryotic host cells by fermentation technology.
- Plasmids are common extrachromosomal genetic elements that replicate independently of the chromosomes of most prokaryotic but also eukaryotic organisms. Cells often have multiple plasmids of different sizes existing together in varying numbers of copies per cell. They are widely used as carriers of genetic information in biological studies involving the analysis of gene function, protein expression or genome editing.
- a custom DNA construct is an artificial segment of nucleic acid created by inserting a target DNA fragment(s) into the backbone of a plasmid vector and is a vehicle for transferring the target DNA fragment(s) into a tissue or cell in order to produce recombinant proteins.
- Recombinant technology plays an irreplaceable role in biotechnology as well as in molecular biology research leading to modem diagnostic and therapeutic approaches.
- Recombinant proteins find their purpose in many applications - in basic research, they are used for studying cellular processes, protein-protein interactions, immune responses and many others. They are also part of common laboratory techniques like ELISA assays, western blots or immunohistochemical methods. Moreover, they indeed are the cornerstones of recombinant vaccines and also find their role in many therapeutics.
- prokaryotic cells is the most widely used among others. In general, lower cost, shorter time, high protein yields and reasonable control in large-scale production are the most prominent advantages.
- the present invention relates generally to an engineered plasmid and selection material for influencing microbial production of useful polypeptides and more particularly to novel plasmid DNA useful in providing exceptionally high levels of exogenous gene expression in E. coli host cells.
- expression vectors with an optimized list of genes exposed to expression in microbial host cell populations such as E. coli.
- the plasmid of the invention is a new artificial highly stable vector with a minimal ORI sequence length with a moderate high copy number creating a minimal burden on the host cell.
- the present invention provides a plasmid vector for protein production using heterologous prokaryotic host cells, such as E. coli.
- the backbone of the plasmid of the invention was derived from the wildtype Corynebacterium renale ORI sequence of the pCR2 replication family. However, the ORI sequence of replication had to be significantly modified to meet the technological demands of recombinant protein production.
- the plasmid of the invention is stable for several passages even without the selection pressure; is particularly suitable for use in bioreactors; enables protein expression at room temperature without the need for excessive heating, aeration nor for extensive stirring; enables overexpression of the produced protein molecule in standard medium (e.g. Luria-Bertani broth); allows to express of proteins that are otherwise difficult to produce.
- standard medium e.g. Luria-Bertani broth
- expression plasmid refers to a plasmid useful for expression of recombinant proteins.
- the most preferred plasmid vector of the invention having SEQ ID NO: 1 is also referred to in this text as “pUbExlOO”.
- the present invention provides an expression vector plasmid of the invention which comprises, in the given order: an origin of replication (ORI sequence); a gene encoding ubiquitin as a leader protein; multiple cloning site (MSC); at least one affinity tag; TEV site; and at least one gene for selection antibiotic resistance; wherein the ORI sequence has a sequence SEQ ID NO: 2:
- Stabilising element is identical to the terminal sequence of the replication initiation protein (RepE protein) gene.
- the newly designed complete ORI sequence (SEQ ID NO: 2) of the plasmid including the stabilizing element is only 755 nucleotides long.
- pBR322 complete ORI sequence of plasmid pET
- pBR322 complete ORI sequence of plasmid pET
- Alteration of replication initiation reduces the amount of biomass required to produce protein units. A decrease in the metabolic activity of biomass also reduces oxygen consumption by approximately 10% (depending on the specific protein produced).
- the plasmid of the invention is stable for several passages even without the selection pressure; is particularly suitable for use in bioreactors; enables protein expression at room temperature without the need for excessive heating, aeration nor for extensive stirring; enables overexpression of the produced protein molecule in standard medium (e.g. Luria-Bertani broth); allows the expression of proteins that are otherwise difficult to produce.
- standard medium e.g. Luria-Bertani broth
- the plasmid of the invention combines the advantages of stable low-copy and high-copy plasmids. Dozens of templates are able to rapidly produce more protein while maintaining template stability.
- the plasmid of the invention has the DNA sequence SEQ ID NO: 1 (and may be referred to further in this text as “pUbExlOO”):
- the plasmid is a double-stranded DNA plasmid.
- SEQ ID NO: 1 represents one strand, and the other strand is complementary to SEQ ID NO: 1.
- the plasmid having the sequence SEQ ID NO: 1 was deposited with the German Collection of Microorganisms and Cell Cultures (Leibniz Institute DSMZ, Inhoffenstr. 7B, D-38124 Braunschweig, Germany) by the Depositor: Veterinary Research Institute, Hudcova 296/70, 62100 Bmo, Czech Republic, under the Accession No. DSM 34045, date of deposit: 19.08.2021.
- the present invention further provides a method of producing a recombinant protein in a heterologous prokaryotic host cell, such as E. coli, said method comprising the steps of: inserting the coding sequence of the protein to be produced into a plasmid according to the invention, transforming the plasmid into a prokaryotic host cell, culturing and harvesting the prokaryotic host cells, disintegrating the prokaryotic host cells to release the produced recombinant protein, optionally purifying the produced recombinant protein and optionally cleaving the leader protein from the produced recombinant protein.
- a heterologous prokaryotic host cell such as E. coli
- the present invention provides a sequence SEQ ID NO: 2, and its use as ORI sequence in plasmids. Detailed Description of the Invention
- the plasmid of the present invention contains, in the listed order: an origin of replication (ORI sequence); a gene encoding ubiquitin as a leader protein; multiple cloning site (MSC); at least one affinity tag; TEV site; and at least one gene for selection antibiotic resistance.
- ORI sequence origin of replication
- MSC multiple cloning site
- TEV site at least one gene for selection antibiotic resistance.
- recombinantly produced protein refers to the protein whose coding sequence is inserted into the plasmid which is in turn transformed into a prokaryotic host cell which then produces the recombinant protein (recombinantly produced protein).
- GOI gene of interest
- Origin of replication (ORI) sequence is a representation of Origin of replication (ORI) sequence:
- plasmids used in the heterologous E. coli expression systems are based on plasmids ColEl, or on closely related pBR322 and pMBl plasmids. Although most origins of replication have the same ORI basis, the resulting plasmids still produce different copy numbers depending on how they are regulated. Generally, replication control is referred to as "relaxed” or “strict” depending on whether the ORI is upregulated by RNA or proteins. A balance between positive and negative regulation results in plasmid copy number range and can be manipulated by mutations in the sequence. For example, pMBl ORI stores about 20 copies per cell, while pUC (pMBl derivative) - which differs by only two mutations - produces up to 700 copies per cell.
- RNA II The prototypical ORI sequence of E. coli, ColEl ORI, which is approximately two kbp in length, is replicated via the Theta mechanism.
- the replication initiation requires the transcription of RNA primer (542 bp), RNA II, from the RNA II promoter (P2).
- RNA II and the P2 promoter are highly conserved across the whole ColEl plasmid family (e.g. DoriC, pCR2, pET28, pIGAL, pBR322, etc.).
- RNA II is necessary to initiate replication because the 3'-end serves as a primer for the DNA polymerase I complex and is essential for DNA replication.
- RNA II activity is mediated by an RNA I molecule (108 bp) that is transcribed from the opposite strand of RNA II from its Pl promoter.
- the ORI sequence (having the sequence SEQ ID NO: 2) is a newly designed minimal origin devised from the pCR2 origin of replication. Only the core region complemented with replication regulating sequence RepE) necessary for replication in E. coli has been preserved in plasmid of the invention.
- the ORI sequence of the plasmid of the invention consists of DNA Unwinding Element (DUE), responsible for opening of the double helix for DNA replication, DNA region coding RNA II and RNA I, promoter (Pl) and Stem sequences which form loops participating in the regulation of the replication.
- DUE DNA Unwinding Element
- Pl promoter
- Stem sequences which form loops participating in the regulation of the replication.
- the essential promoter P2 critical for RNA II primer transcription, was replaced by de-novo designed promoter.
- the regulation DNA hairpin was replaced by a DNA sequence that cannot fold into a secondary structure element. Moreover, the high AT pair-rich region was utterly redesigned. Removing translation signal hairpin and implementing promotor with low affinity to RNA polymerase results in stable low-speed replication with medium -range copy number of the plasmid of the invention in E. coli host cells.
- Plasmid of the invention contains in its complete ORI sequence a newly designed P2 promoter and a stabilizing element, which is a sequence identical to the terminal sequence of the replication initiation protein (terminal amino acid sequence of the RepE protein).
- the RepE protein plays an essential role in initiating replication from the origin, ori2.
- the RepE protein has two main functions: initiation of replication from the origin, ori2, and autogenous repression of repE transcription.
- Monomers of RepE represent the active form by binding to ori2 to initiate replication, while dimers act as an autogenous repressor by binding to the operator.
- Increased expression of the RepE gene switches the initiation of replication to autogenous repression, which leads to a reduction in replication and thus to a reduction in the number of plasmid copies.
- the stabilizing element regulates the number of RepE protein molecules so that the replication of the plasmid is not blocked. This occurs through hybridization of the antisense RNA with the complementary region of the gene for the RepE protein.
- the plasmid of the invention was tested for stability in two host strains, DH10 ⁇ and BL21 (DE3). In both host strains, it showed a comparable stability to the reference plasmid pET28 (Fig. 1) at a one third of the plasmid size.
- the ubiquitin (Ub) gene encodes a protein that interacts with most other proteins with a Kdd affinity > 0.3 mM, which obscures the hydrophobic segments of the fusion protein with a direct effect on protein expression and solubility.
- ubiquitin was an eukaryotic 8.5 kDa protein with many functions. Its sequence is highly conserved from unicellular organisms to humans. Its presence in the sequence facilitates the production, isolation and identification of the protein from the host production system. Ubiquitin also increases the solubility of the fusion protein produced (Rogov VV, Rozenknop A, Rogova NY, Lohr F, Tikole S, Jaravine V, Gimtert P, Dikic I, Dotsch V.
- the gene encoding ubiquitin is optimized for codon usage in E. colt and preferably has a DNA sequence SEQ ID NO: 3. This gene sequence corresponds to the amino acid sequence SEQ ID NO: 4.
- MCS Multiple cloning site
- the DNA sequence for a recombinantly produced protein is inserted into the multiple cloning site (MCS) of the expression vector.
- MCS multiple cloning site
- the multiple cloning site or polylinker forms a short DNA segment (SEQ ID NO: 5) that contains a series of 8 restriction enzyme sites (RE) which are sites for Kpnl, Ncol, BamHI, PstI, SacI, Hindlll, Notl, Sall.
- the expression vector of the invention contains nucleotide sequences encoding peptide tag enabling detection and purification of recombinant protein product out of the crude biological source by an affinity technique.
- the plasmid of the invention preferably contains two versions of polyhistidine tags in its sequence, a 14xHisTag and a 6xHisTag.
- a peptide tag containing 14 histidines (14xHisTag) and additional amino acids (G, S and T) forming loops in its secondary structure is attached on the N-terminus of a recombinantly produced protein. Due to its structure, the 14xHisTag elicits higher affinity to divalent cations and therefore may improve the purification process by metallochelating methods.
- Proteins expressed from the vector plasmid of the invention have the ability to cleave the 14xHisTag after purification due to the presence of TEV cleavage site in between of a tag sequence and the protein of interest.
- the 14xHisTag DNA sequence is preferably SEQ ID NO: 6, and the corresponding amino acid sequence is SEQ ID NO: 7.
- the expression vector of the invention also contains a conventional 6xHisTag sequence behind the multiple cloning site (MCS), which can be used as a C-terminal peptide tag for the metallochelation carrier.
- MCS multiple cloning site
- 6xHisTag in a protein sequence may improve detection of a protein by commercially available specific anti-HisTag antibody.
- the gene of interest GOI, gene encoding the recombinant protein to be produced
- GOI gene encoding the recombinant protein to be produced
- sequence encoding 6xHisTag is SEQ ID NO: 8, and the corresponding amino acid sequence is SEQ ID NO: 9.
- TEV site is a short amino acid sequence enabling the cleavage of ubiquitin sequence and 14xHisTag from the recombinantly produced protein by a TEV protease.
- TEV cleavage site is in the plasmid of the invention encoded by a DNA sequence SEQ ID NO: 10, and the corresponding amino acid sequence is SEQ ID NO: 11.
- the preferred selection antibiotic is kanamycin
- the preferred selection antibiotic resistance gene is a codon optimised kanamycin resistance gene having the sequence SEQ ID NO: 12.
- the kanamycin resistance gene Km R which is used in a number of E. coli expression plasmids, including pET plasmids, was originally isolated from transposable element Tn903 in E. coli. This gene encodes an aminoglycoside 3 '-phosphotransferase (kanamycin kinase; neomycin-kanamycin phosphotransferase). Aminoglycoside phosphotransferases inactivate aminoglycoside antibiotics through phosphorylation. The original gene had a CAI (Codon adaptation index) of 0.39 and was improved to 0.95 after codon usage optimization.
- the kanamycin resistance gene sequence is preferably SEQ ID NO: 12, its complementary sequence is SEQ ID NO: 13, and the corresponding amino acid sequence is SEQ ID NO: 14.
- Figtire 1 The plasmid of the invention stability in E. coli BL21 (DE3) cells - a comparative study of plasmid retention under non-selective conditions for four subsequent passages. Bars indicate optical density after 24 h of cultivation with and without the addition of antibiotics. On the left side, results for cells transformed with the plasmid having SEQ ID NO: 1 are depicted. On the right side, results of control cells transformed with pET28 are depicted.
- the plasmid map comprises the DNA sequence of SEQ ID NO: 2 encoding ORI gene, the ubiquitin leader protein of SEQ ID NO: 3, 14xHisTag sequence for affinity purification (SEQ ID NO: 6) and optimized gene for kanamycin resistance (SEQ ID NO: 12).
- the DNA sequence for the gene of interest can be inserted into the multiple cloning site (MCS - (SEQ ID NO: 5) of the expression vector plasmid of the invention.
- MCS - multiple cloning site or polylinker forms a short segment of DNA that contains a series (generally up to 8 REs).
- Restriction enzyme (RE) sites - are Kpnl, Ncol, BamHI, PstI, Sad, Hindlll, Notl, Sall. Examples
- a plasmid according to the invention having SEQ ID NO: 1 (plasmid pUbExlOO) was created by design de novo synthetically.
- the linear plasmid was circularized by ligation and, after transformation into the target host cells, was propagated in them and subsequently isolated.
- the DNA sequence for a recombinantly produced protein is inserted into the multiple cloning site (MCS) of the expression vector plasmid having the sequence SEQ ID NO: 1 (plasmid pUbExlOO).
- MCS multiple cloning site
- the multiple cloning site is formed by a short DNA segment that contains a series of short DNA sequences recognised by restriction enzymes in following order Kpnl, Ncol, BamHI, PstI, Sad, Hindlll, Notl, Sall.
- a cleavage site may be included between the leader protein Ubq with 14xHis-Tag peptide for IMAC affinity purification (Immobilized Metal Chelate Affinity Chromatography) and the protein of interest to cleave off the leader peptide and purify recombinant protein.
- the gene of interest may be inserted in any of the cleavage sites in the MCS. All genes were codon optimised and shared common restriction sites at 5’- and 3’- ends of coding sequences. The incorporation procedure, as well as protein expression experiments, were done under the same conditions.
- Gene for the recombinantly produced protein was processed by restriction enzymes Ncol/Notl in CutSmart restriction Buffer (New England Biolabs, USA) for 1 hour at 37°C.
- the restriction mixture was separated by DNA electrophoresis.
- the DNA fragment of appropriate size was excised and isolated by isolation kit NucleoSpin Gel and PCR Clean-up (Macherey-Nagel). The DNA concentration was determined spectrophotometrically.
- the vector was linearised by the same set of restriction enzymes. The incorporation of the gene of interest was achieved via DNA ligation. Ten ng of the insert was incubated with 100 ng of linearised vector plasmid of the invention in the presence of T4 DNA ligase for 12 hours at 16°C.
- the reaction product was used for heat-shock transformation of E. coli DHIOp chemo-competent cells. Cultivation on agar plates with kanamycin selection was used to select cells carrying the plasmid. The presence of the gene of interest in the plasmid was confirmed by DNA sequencing.
- Plasmid transformation Vector plasmid of the invention containing the gene of interest coding sequence was used to transform production strain E. coli BL21 (DE3). 50 ng of plasmid DNA was added to chemo -competent cells, which were transformed by heat shock (42° C/60 sec.). Positive clones were selected using seeding on LB agar plates with kanamycin. Fifteen colonies from the Petri dish were transferred to a liquid LB medium with kanamycin (100 pg/ml) and glucose (1 %).
- the inoculum was cultivated at 37°C for 16 hours under constant agitation at 220 rpm.
- 1 ml of cell culture was used to inoculate 100 ml of sterile autoinduction media (composition for IL: 10 g Tryptone; 5 g Yeast extract; 3.3 g ammonium sulphate; 50 mM phosphate buffer pH 6.7; 0.5 g glucose; 2 g lactose).
- composition for IL 10 g Tryptone; 5 g Yeast extract; 3.3 g ammonium sulphate; 50 mM phosphate buffer pH 6.7; 0.5 g glucose; 2 g lactose.
- the cultivation was kept for 48 hours at 22°C under constant agitation at 220 rpm.
- the cultivation was terminated by cooling down the cell suspension to 4°C.
- the cells were harvested by centrifugation (6000 g/4°C/10 min.).
- the bacterial pellet was resuspended in the appropriate buffer (e.g. 100 mM Tris-HCl pH 8, 300 mM NaCl, 0.1 % Tween 20 (v/v), 10 mM Imidazole) and subsequently disintegrated by high pressure homogenisation.
- Homogenised cell suspension was centrifuged for one hour at 4°C and 14000 g, fdtrated through 0.22 pm fdter and loaded in the same buffer to IMAC column.
- Non-specifically bound proteins were washed by increasing the concentration of imidazole to 125 mM.
- the recombinantly produced protein was eluted by 250 mM imidazole in the same buffer.
- the TEV protease processing can be used for leader protein removal.
- Purified protein was dialysed against the same buffer as used for purification except the imidazole (e.g. 100 mM Tris-HCl pH 8, 300 mM NaCl, 0.1 % Tween 20 (v/v)). Dialysed protein was incubated with TEV protease in the 1:30 ratio (w/w) for 16 hours at 4°C. Cleaved mixture was loaded on the IMAC column and the unbound protein (where the leader protein including his-tag was cleaved) is collected.
- imidazole e.g. 100 mM Tris-HCl pH 8, 300 mM NaCl, 0.1 % Tween 20 (v/v)
- the stability of the plasmid having the sequence SEQ ID NO: 1 in E. coli genome was verified.
- the test for plasmid presence was performed as follows. 10 pl of liquid culture was used to inoculate 10 ml of liquid broth with antibiotics, and 20 pl was seeded onto the agar plate with antibiotics. The optical density and colony counting were performed after 16 h of cultivation at 37°C. Five subsequent passages were done, and the colony counting, and optical density measurements were performed for each of them.
- the novel plasmid of the invention is cost-effective and can reduce the use of antibiotics in large-scale protein production.
- a combination of plasmid DNA isolation methods, cell number counting and quantitative polymerase chain reaction (qPCR) was used to determine the copy number of plasmid DNA in the low copy, medium copy or high copy plasmid range. Copy number was determined against standard high copy (pUC57) and low copy (pET28) plasmids. All tests described below were performed in technically and biologically independent triplicates.
- the growth rate of A. coll DHIOp chemically competent cells transformed with plasmids pUC57, pET28 or the plasmid of the invention, in which marker gene for lysostaphin protein was cloned was determined.
- Heat-shock transformation in KCM buffer and subsequent cultivation on microbiological LB agar with the addition of suitable antibiotics - kanamycin for pET28 and of the invention, and ampicillin for pUC57 were used for the transformation.
- Cultivation was performed at 37°C for 16 hours. Three colonies were randomly selected from each plate and inoculated into a liquid LB medium with a suitable antibiotic (ampicillin or kanamycin). Cultivation was continued with stirring (180 rpm) at 37°C for 16 hours.
- plasmid DNA isolations for all described cultures were done using EZNA Plasmid Mini Kit 1 (Omega Bio-Tek). The isolation procedure was performed according to the manufacturer's instructions. 4 ml of cell suspension from each culture (9 plasmid DNA isolations) were used as input material. The isolated plasmid DNA was eluted with 60 pl of elution buffer, and the plasmid DNA concentration was determined spectrophotometrically. The isolated plasmid DNA was serially diluted for each of the samples in demineralized water in a quality suitable for PCR at a decimal dilution from 10 1 - 10 12 . The qPCR amplification Ct value was determined for all these samples.
- the primers used for amplification are identical to the primers used for amplification:
- Reverse primer (-) SEQ ID NO: 17: 5 ' - TCTCGCTTTTATACAGGGTG - 3 '
- Both primers define a specific binding region of 248 base pairs in the gene encoding the lysostaphin protein.
- the reaction temperature profile was composed of an initial denaturation of 95°C for 10 min, 45 cycles alternating 95°C for 10 s, 60°C for 20 s and 72°C for 20 s.
- the melting point of the PCR products was analysed with an increment of 0.5 °C in the range of 60-95 °C for 30 minutes. Samples were plated in duplicate for all DNA dilutions, for a total of 9 x 240 samples. PCR assay and melting point analysis were performed by detecting a change in fluorescence in the reaction mixture containing SybrGreen intercalation dye.
- Plasmid DNA copy number was used based on pET28 low copy ( ⁇ 15 - 20) and pUC57 high copy (-500- 700) standards in cases where at all dilutions the melting point analysis corresponded to a single PCR product of 82.5°C, which corresponds to the expected product.
- Apxla is related to RTX toxins (APXI - APXIV) of Actinobacilus pleuropneumonia.
- APXla is a strongly cytotoxic and haemolytic pore-forming factor. Together with capsular polysaccharides and mural lipo-polysaccharides, Apxla is an essential factors of virulence and thus responsible for fibrinhaemorrhagic pleuropneumonia in swine.
- the DNA sequence coding for Apxla C -terminal domain sequence with E. colt optimized codon usage distribution was synthesized and inserted into the expression vector via restriction enzyme digestion and subsequent ligation. The produced protein is of 27 kDa apparent molecular weight.
- Plasmid of the invention with an inserted Apxla gene (SEQ ID NO: 18):
- Gene encoding Apxla was processed by restriction enzymes Ndel/Notl in CutSmart restriction Buffer (New England Biolabs, USA) for 1 hour at 37°C.
- the restriction mixture was separated using DNA electrophoresis.
- the DNA fragment of appropriate size was excised and isolated by isolation kit NucleoSpin Gel and PCR Clean-up (Macherey-Nagel). The DNA concentration was determined spectrophotometrically.
- the vector was linearised by the same procedure. The incorporation of the gene of interest was achieved via DNA ligation. 10 ng of the linearised insert was incubated with 100 ng of linearised vector plasmid of the invention in the presence of T4 DNA ligase for 12 hours at 16°C. The reaction product was used for heat-shock transformation of E.
- Vector plasmid of the invention containing the gene of interest coding sequence was used to transform production strain E. coli BL21 (DE3). 50 ng of plasmid DNA was added to chemo -competent cells, which were transformed by heat shock (42°C/60 sec.) in the water bath. Positive clones were selected using seeding on LB agar plates with kanamycin. Fifteen colonies from the Petry dish were transferred to a liquid LB medium with kanamycin (100 pg/ml) and glucose (1%). The inoculum was cultivated at 37°C for 12 hours under constant agitation at 220 rpm.
- 1 ml of cell culture was used to inoculate 100 ml of sterile autoinduction media (composition for IL: 10 g Tryptone; 5 g Yeast extract; 3.3 g ammonium sulphate; 50 mM phosphate buffer pH 6.7; 0.5 g glucose; 2 g lactose).
- composition for IL 10 g Tryptone; 5 g Yeast extract; 3.3 g ammonium sulphate; 50 mM phosphate buffer pH 6.7; 0.5 g glucose; 2 g lactose.
- the cultivation was kept for 48 hours at 22°C under constant agitation at 220 rpm.
- the protein production was verified by protein SDS- PAGE electrophoresis and subsequent Coomasie Brilliant blue staining of whole cell lysates. For three proteins, the production rate was analysed in more detail.
- Plasmid pUbExlOO improves the subsequent purification process and makes its production much more efficient.
- Bst 5.9 polymerase is a chimeric enzyme consisting of the main scaffold of Bst polymerase from Thermus aquations Taq polymerase. A short loop from Bst polymerase from Geobacillus stearothermophilus was introduced. Bst 5.9 polymerase is used in the isothermal amplification reaction. The coding sequence with E. coli optimized codon usage distribution was synthesized and inserted into the expression vector via restriction enzyme digestion and subsequent ligation. The produced protein is of 72 kDa apparent molecular weight.
- Plasmid of the invention with an inserted Bst 5.9 gene (SEQ ID NO: 19)
- Gene of interest was processed by restriction enzymes Ndel/Notl in CutSmart restriction Buffer (New England Biolabs, USA) for 1 hour at 37°C.
- the restriction mixture was separated using DNA electrophoresis.
- the DNA fragment of appropriate size was excised and isolated by isolation kit NucleoSpin Gel and PCR Clean-up (Macherey-Nagel). The DNA concentration was determined spectrophotometrically.
- the vector plasmid of the invention was linearised by the same set of restriction enzymes and the same procedure. The incorporation of the gene of interest was achieved via DNA ligation. Ten ng of the linearised insert was incubated with 100 ng of linearised vector plasmid of the invention in the presence of T4 DNA ligase for 12 hours at 16°C.
- the reaction product was used for heat-shock transformation of E. coli DH10 ⁇ chemo-competent cells. Cultivation on agar plates with kanamycin selection was used to select cells carrying the plasmid. The presence of the gene of interest in the vector plasmid of the invention was confirmed by DNA sequencing.
- Vector plasmid of the invention containing the gene of interest coding sequence was used to transform production strain E. coli BL21 (DE3). 50 ng of plasmid DNA was added to chemo -competent cells, which were transformed by heat shock (42°C/60 sec.) in the water bath. Positive clones were selected using seeding on LB agar plates with kanamycin. Fifteen colonies from the Petri dish were transferred to a liquid LB medium with kanamycin (100 pg/ml) and glucose (1 %). The inoculum was cultivated at 37°C for 12 hours under constant agitation at 220 rpm.
- 1 ml of cell culture was used to inoculate 100 ml of sterile autoinduction media (composition for IL: 10 g Tryptone; 5 g Yeast extract; 3.3 g ammonium sulphate; 50 mM phosphate buffer pH 6.7; 0.5 g glucose; 2 g lactose).
- composition for IL 10 g Tryptone; 5 g Yeast extract; 3.3 g ammonium sulphate; 50 mM phosphate buffer pH 6.7; 0.5 g glucose; 2 g lactose.
- the cultivation was kept for 48 hours at 22°C under constant agitation at 220 rpm.
- the protein production was verified by protein SDS- PAGE electrophoresis and subsequent Coomasie Brilliant blue staining of whole cell lysates. For three proteins, the production rate was analysed in more detail.
- Bst 5.9 protein was compared with the commonly used laboratory expression plasmid pET.
- the difference is most pronounced in the first 24 hours, when the plasmid of the invention-Bst5.9 has a density of only 3.5 McFarland and pET28-Bst5.9 has a density of 25 McFarland.
- the density of pET28-Bst5.9 increased to 35 McFarland and pUbExlOO-Apxla to 42 McFarland.
- the resulting pUbExl00-Bst5.9 pellet had a higher yield (14.5 g/L) than pET28-Bst5.9 (8.75 g/L).
- the final protein production was 3.3-fold higher with the new plasmid of the invention-Apxla. Due to the relatively higher production of protein per unit of biomass, the plasmid of the invention improves the subsequent purification process and makes its production significantly more efficient. Production of lysostaphin using the plasmid of the invention.
- Lysostaphin is a 27 kDa metalloendopeptidase, the antimicrobial lytic enzyme that is produced by Staphylococcus simulans. Lysostaphin is highly active against Staphylococcus aureus strains. The bacteriolytic activity of lysostaphin is due to its ability to hydrolyse the penta-glycine cross bridge of .S' aureus peptidoglycan. The coding sequence with E. coli-optimized codon usage distribution was synthesized and inserted into the expression vector via restriction enzyme digestion and subsequent ligation.
- Plasmid of the invention with an inserted Lysostaphine gene (SEQ ID NO: 20):
- Gene for lysostaphin was processed by restriction enzymes Ndel/Notl in CutSmart restriction Buffer (New England Biolabs, USA) for 1 hour at 37°C.
- the restriction mixture was separated using DNA electrophoresis.
- the DNA fragment of appropriate size was excised and isolated by isolation kit NucleoSpin Gel and PCR Clean-up (Macherey-Nagel). The DNA concentration was determined spectrophotometrically.
- the vector was linearised by the same procedure. The incorporation of the gene of interest was achieved via DNA ligation. Ten ng of the linearised insert was incubated with 100 ng of linearised vector plasmid of the invention in the presence of T4 DNA ligase for 12 hours at 16°C. The reaction product was used for heat-shock transformation of E.
- coli DH10 ⁇ chemo-competent cells Cultivation on agar plates with kanamycin selection was used to select cells carrying the plasmid. The presence of the gene of interest in the plasmid of the invention was confirmed by DNA sequencing.
- the plasmid of the invention containing lysostaphin gene coding sequence was used to transform production strain E. colt BL21 (DE3). 50 ng of plasmid DNA was added to chemo -competent cells, which were transformed by heat shock (42°C/60 sec.) in the water bath. Positive clones were selected using seeding on LB agar plates with kanamycin. Fifteen colonies from the Petri dish were transferred to a liquid LB medium with kanamycin (100 pg/ml) and glucose (1 %). The inoculum was cultivated at 37°C for 12 hours under constant agitation at 220 rpm.
- 1 ml of cell culture was used to inoculate 100 ml of sterile autoinduction media (composition for IL: 10 g Tryptone; 5 g Yeast extract; 3.3 g ammonium sulphate; 50 mM phosphate buffer pH 6.7; 0.5 g glucose; 2 g lactose).
- composition for IL 10 g Tryptone; 5 g Yeast extract; 3.3 g ammonium sulphate; 50 mM phosphate buffer pH 6.7; 0.5 g glucose; 2 g lactose.
- the cultivation was kept for 48 hours at 22°C under constant agitation at 220 rpm.
- the protein production was verified by protein SDS- PAGE electrophoresis and subsequent Coomasie Brilliant blue staining of whole cell lysates. For three proteins, the production rate was analysed in more detail. Comparison of expression of lysostaphin.
- lysostaphin protein was compared with the commonly used laboratory expression plasmid pET.
- the difference is most pronounced in the first 24 hours, when of the invention-Lysostaphin has a density of only 5 McFarland and pET28-Lysostaphin has a density of 13 McFarland.
- the density of pET28- Lysostaphin increased to 22 McFarland and of the invention-Lysostaphin to 25 McFarland.
- the resulting plasmid of the invention-Lysostaphin pellet had a lower yield (8.25 g/L) than pET28- Lysostaphin (13.75 g/L). However, the final protein production was 4.96x higher with the pUbExlOO- Lysostaphin. Due to the relatively higher production of protein per unit of biomass, the plasmid of the invention improves the subsequent purification process and makes its production significantly more efficient.
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Plant Pathology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Saccharide Compounds (AREA)
Abstract
The present invention provides a novel expression vector (plasmid) containing a newly designed ORI sequence. The novel expression vector is more stable and provides for a high production of recombinant protein per total biomass.
Description
EXPRESSION VECTOR FOR PRODUCTION OF RECOMBINANT PROTEINS IN
PROKARYOTIC HOST CELLS
Field of Art
The present invention provides a novel expression vector particularly suitable for the production of recombinant proteins in prokaryotic host cells by fermentation technology.
Background Art
Plasmids are common extrachromosomal genetic elements that replicate independently of the chromosomes of most prokaryotic but also eukaryotic organisms. Cells often have multiple plasmids of different sizes existing together in varying numbers of copies per cell. They are widely used as carriers of genetic information in biological studies involving the analysis of gene function, protein expression or genome editing. A custom DNA construct is an artificial segment of nucleic acid created by inserting a target DNA fragment(s) into the backbone of a plasmid vector and is a vehicle for transferring the target DNA fragment(s) into a tissue or cell in order to produce recombinant proteins.
In the last few decades, recombinant technology plays an irreplaceable role in biotechnology as well as in molecular biology research leading to modem diagnostic and therapeutic approaches. Recombinant proteins find their purpose in many applications - in basic research, they are used for studying cellular processes, protein-protein interactions, immune responses and many others. They are also part of common laboratory techniques like ELISA assays, western blots or immunohistochemical methods. Moreover, they indeed are the cornerstones of recombinant vaccines and also find their role in many therapeutics. There are several ways of producing recombinant proteins, however, using prokaryotic cells is the most widely used among others. In general, lower cost, shorter time, high protein yields and reasonable control in large-scale production are the most prominent advantages.
Summary of the Invention
The present invention relates generally to an engineered plasmid and selection material for influencing microbial production of useful polypeptides and more particularly to novel plasmid DNA useful in providing exceptionally high levels of exogenous gene expression in E. coli host cells. There are currently many examples of expression vectors with an optimized list of genes exposed to expression in microbial host cell populations such as E. coli.
The plasmid of the invention is a new artificial highly stable vector with a minimal ORI sequence length with a moderate high copy number creating a minimal burden on the host cell.
The present invention provides a plasmid vector for protein production using heterologous prokaryotic host cells, such as E. coli. The backbone of the plasmid of the invention was derived from the wildtype Corynebacterium renale ORI sequence of the pCR2 replication family. However, the ORI sequence of replication had to be significantly modified to meet the technological demands of recombinant protein production.
The plasmid of the invention is stable for several passages even without the selection pressure; is particularly suitable for use in bioreactors; enables protein expression at room temperature without the need for excessive heating, aeration nor for extensive stirring; enables overexpression of the produced protein molecule in standard medium (e.g. Luria-Bertani broth); allows to express of proteins that are otherwise difficult to produce.
The terms “expression plasmid”, “plasmid”, “expression vector plasmid”, “plasmid vector” and “expression vector” are used in this text interchangeably, and all refer to a plasmid useful for expression of recombinant proteins. The most preferred plasmid vector of the invention having SEQ ID NO: 1 is also referred to in this text as “pUbExlOO”.
The present invention provides an expression vector plasmid of the invention which comprises, in the given order: an origin of replication (ORI sequence); a gene encoding ubiquitin as a leader protein; multiple cloning site (MSC); at least one affinity tag; TEV site; and at least one gene for selection antibiotic resistance; wherein the ORI sequence has a sequence SEQ ID NO: 2:
5 ' -GCAGACAGTTTTATTGTTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGT AGAAAAGAGCGCATGCCGTGAGACTGCGGCGGGCGTTACCGGCTCACAAATAACGGGATACGCAGGCAGTGCTCA AATCAGGAAGGACCGGAAAAAGGATGCGGCGTAGCCGTTTTTCCATAGGCTCCGCCCCCCTGACAAGCATCACGA AATCTGACGCTCAAATCAGTGGCGGCGAAACCCGACAGGACTATAAAGATCCCAGGCGTTTCCCCCTGGTAGCTC CCTCGTGCGCTCTCCTGTTCCTGCCTTTCGGTTTACCGGTGTCATTCCGCTGTTATGGCCGCGTTTGTCTCATTC CACGCCTGACACTCAGTTCCGGGTAGGCAGTTCGCTCCAAGCTGGACTGTATGCACGAACCCCCCGTTCAGTCCG ACTACCACGCCCGTTCCGGTAACTATCAACTTGAGTCCAACCCGGAAAGACACGACAAATCGCCAGTGGCGGTAG CCATTGGTAACTGAGATGTGCGAGAGATTTATCTGGAGTTCTTGAAGTGGGGGCCTGAGTGCGGCTACACTGGAA GGACAGTTTAGGTGACTCGTCTCGCACAAGACAGTTACCACGGTTCATAAACTTGCCGGTTTAATGAACCTTCGA AAAACCACCTTGCCGGGTGGTTTTTTCTTTTCAAAGAAGATACGCGTTTACTCTTCGCCTCGTCAATGAAGGGTC GCATTAAT-3 '
The complete ORI sequence newly designed for the plasmid of the invention was derived from the pCR2 ORI sequence, where the essential P2 promoter, critical for RNA II primer transcription, was replaced by a de-novo designed promoter and a stabilizing element (86 bp long sequence, underlined in the SEQ ID NO: 2 above) was added to the complete sequence. Stabilising element is identical to the terminal sequence of the replication initiation protein (RepE protein) gene. The newly designed complete ORI sequence (SEQ ID NO: 2) of the plasmid including the stabilizing element is only 755 nucleotides long. Compared to the complete ORI sequence of plasmid pET (pBR322), which is 1230 nucleotides long, it constitutes only 61% of the length of the complete pET ORI sequence. Of these 755 nucleotides, only 418 nucleotides are identical to the core ORI sequence of plasmid pET (56%). Alteration of replication initiation reduces the amount of biomass required to produce protein units. A decrease in the metabolic activity of biomass also reduces oxygen consumption by approximately 10% (depending on the specific protein produced).
The plasmid of the invention is stable for several passages even without the selection pressure; is particularly suitable for use in bioreactors; enables protein expression at room temperature without the need for excessive heating, aeration nor for extensive stirring; enables overexpression of the produced protein molecule in standard medium (e.g. Luria-Bertani broth); allows the expression of proteins that are otherwise difficult to produce.
The plasmid of the invention combines the advantages of stable low-copy and high-copy plasmids. Dozens of templates are able to rapidly produce more protein while maintaining template stability.
In one particularly preferred embodiment, the plasmid of the invention has the DNA sequence SEQ ID NO: 1 (and may be referred to further in this text as “pUbExlOO”):
5 ' -CTCGAGTCTGCCCACCACCACCACTCTGGTCACCACCACACGGGTCACCACCACCACTCTGGTTCTCACCAC CACTCTGGTGCCGAAAACCTGTACTTCCAGTCTGGTTCTGGTACCATGGTTGGATCCGCTGCAGGAGCTCAAGCT TGCGGCCGCAGTCGACCACCACCACCACCACCACTGAGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTT GGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCT GAAAGGAGGAACTATATCCGGATGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCT CATGAATTAATTCTTAGAAAAACTCATCCAGCATCAGGTGAAATTGCAGCTTATTCATATCCGGGTTGTCGATGC CATATTTTTGAAACAGACGCTTTTGCAGGCTCGGGCTGAACTCGCCCAGGCAGTTCCACAGAATCGCCAGATCTT GGTAACGGTCCGCGATGCCAACACGACCCACATCAATGCAGCCGATCAGTTTACCCTCGTCAAAGATCAGGTTAT CCAGGCTGAAGTCGCCGTGGGTAACCACGCTATCCGGGCTAAACGGCAGCAGTTTGTGCATTTCCTTCCAAACCT GCTCCACCGGCCAGCCGTTACGTTCATCGTCGAAATCGCTCGCGTCAACCAGACCGTTGTTCATACGGCTTTGCG CCTGCGCCAGACGAAAAACACGGTCGCTGTTGAACGGGCAGTTGCACACCGGAATGCTGTGCAGACGACGCAGAA AAACCGCCAGCGCATCCACGATGTTCTCGCCGCTGTCCGGGTATTCCTCCAGCACTTGGAACGCGGTTTTACCCG GAATCGCGGTGGTCAGCAGCCACGCATCATCCGGGGTACGAATAAAGTGCTTGATGGTCGGCAGCGGCATGAACT
CGGTCAGCCAGTTCAGACGAACCATCTCATCGGTCACGTCGTTCGCCACGCTGCCTTTACCGTGCTTCAGGAACA GCTCCGGCGCATCCGGCTTGCCATACAGACGGTAGATGGTCGCGCCGCTCTGACCCACGTTGTCACGCGCCCACT TATAACCATACAGATCCGCATCCATGTTGCTATTCAGACGCGGACGGCTACAGCTGGTCTCACGTTGAATGTGCA TAACACCCCTTGTATTACTGTTTATGTAAGCAGACAGTTTTATTGTTCATGACCAAAATCCCTTAACGTGAGTTT TCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGAGCGCATGCCGTGAGACTGCGGCGGGCGTTACCGGCTCACAA ATAACGGGATACGCAGGCAGTGCTCAAATCAGGAAGGACCGGAAAAAGGATGCGGCGTAGCCGTTTTTCCATAGG CTCCGCCCCCCTGACAAGCATCACGAAATCTGACGCTCAAATCAGTGGCGGCGAAACCCGACAGGACTATAAAGA TCCCAGGCGTTTCCCCCTGGTAGCTCCCTCGTGCGCTCTCCTGTTCCTGCCTTTCGGTTTACCGGTGTCATTCCG CTGTTATGGCCGCGTTTGTCTCATTCCACGCCTGACACTCAGTTCCGGGTAGGCAGTTCGCTCCAAGCTGGACTG TATGCACGAACCCCCCGTTCAGTCCGACTACCACGCCCGTTCCGGTAACTATCAACTTGAGTCCAACCCGGAAAG ACACGACAAATCGCCAGTGGCGGTAGCCATTGGTAACTGAGATGTGCGAGAGATTTATCTGGAGTTCTTGAAGTG GGGGCCTGAGTGCGGCTACACTGGAAGGACAGTTTAGGTGACTCGTCTCGCACAAGACAGTTACCACGGTTCATA AACTTGCCGGTTTAATGAACCTTCGAAAAACCACCTTGCCGGGTGGTTTTTTCTTTTCAAAGAAGATACGCGTTT ACTCTTCGCCTCGTCAATGAAGGGTCGCATTAATTCGATCCCGCGAAATTAATACGACTCACTATAGGGGAATTG TGAGCGGATAACAATTCCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACATATGCAGATCTTCG TTAAAACCCTGACCGGTAAAACCATCACCCTGGAAGTTGAACCGTCTGACACCATCGAAAACGTTAAAGCGAAAA TCCAGGACAAAGAAGGTATCCCGCCGGACCAGCAGGAACTGATCTTCGCGGGTAAACAGCTGGAAGACGGTCGTA CCCTGTCTGACTACAACATCCAGAAAGAATCTACCCTGCACCTGGTTCTGCAG- 3 '
The plasmid is a double-stranded DNA plasmid. SEQ ID NO: 1 represents one strand, and the other strand is complementary to SEQ ID NO: 1.
The plasmid having the sequence SEQ ID NO: 1 was deposited with the German Collection of Microorganisms and Cell Cultures (Leibniz Institute DSMZ, Inhoffenstr. 7B, D-38124 Braunschweig, Germany) by the Depositor: Veterinary Research Institute, Hudcova 296/70, 62100 Bmo, Czech Republic, under the Accession No. DSM 34045, date of deposit: 19.08.2021.
In another aspect, the present invention further provides a method of producing a recombinant protein in a heterologous prokaryotic host cell, such as E. coli, said method comprising the steps of: inserting the coding sequence of the protein to be produced into a plasmid according to the invention, transforming the plasmid into a prokaryotic host cell, culturing and harvesting the prokaryotic host cells, disintegrating the prokaryotic host cells to release the produced recombinant protein, optionally purifying the produced recombinant protein and optionally cleaving the leader protein from the produced recombinant protein.
In yet another aspect, the present invention provides a sequence SEQ ID NO: 2, and its use as ORI sequence in plasmids.
Detailed Description of the Invention
The plasmid of the present invention contains, in the listed order: an origin of replication (ORI sequence); a gene encoding ubiquitin as a leader protein; multiple cloning site (MSC); at least one affinity tag; TEV site; and at least one gene for selection antibiotic resistance.
The term “recombinantly produced protein” refers to the protein whose coding sequence is inserted into the plasmid which is in turn transformed into a prokaryotic host cell which then produces the recombinant protein (recombinantly produced protein).
The term “gene of interest” (GOI) is the coding sequence of the recombinantly produced protein which is inserted into the plasmid.
Origin of replication (ORI) sequence:
Most plasmids used in the heterologous E. coli expression systems are based on plasmids ColEl, or on closely related pBR322 and pMBl plasmids. Although most origins of replication have the same ORI basis, the resulting plasmids still produce different copy numbers depending on how they are regulated. Generally, replication control is referred to as "relaxed" or "strict" depending on whether the ORI is upregulated by RNA or proteins. A balance between positive and negative regulation results in plasmid copy number range and can be manipulated by mutations in the sequence. For example, pMBl ORI stores about 20 copies per cell, while pUC (pMBl derivative) - which differs by only two mutations - produces up to 700 copies per cell.
The prototypical ORI sequence of E. coli, ColEl ORI, which is approximately two kbp in length, is replicated via the Theta mechanism. The replication initiation requires the transcription of RNA primer (542 bp), RNA II, from the RNA II promoter (P2). RNA II and the P2 promoter are highly conserved across the whole ColEl plasmid family (e.g. DoriC, pCR2, pET28, pIGAL, pBR322, etc.). RNA II is necessary to initiate replication because the 3'-end serves as a primer for the DNA polymerase I complex and is essential for DNA replication. RNA II activity is mediated by an RNA I molecule (108 bp) that is transcribed from the opposite strand of RNA II from its Pl promoter.
The ORI sequence (having the sequence SEQ ID NO: 2) is a newly designed minimal origin devised from the pCR2 origin of replication. Only the core region complemented with replication regulating sequence RepE) necessary for replication in E. coli has been preserved in plasmid of the invention. The ORI sequence of the plasmid of the invention consists of DNA Unwinding Element (DUE), responsible for opening of the double helix for DNA replication, DNA region coding RNA II and RNA I, promoter (Pl) and Stem sequences which form loops participating in the regulation of the replication. The essential promoter P2, critical for RNA II primer transcription, was replaced by de-novo designed
promoter. Compared to other ColEl family origins used in protein production vectors, the regulation DNA hairpin was replaced by a DNA sequence that cannot fold into a secondary structure element. Moreover, the high AT pair-rich region was utterly redesigned. Removing translation signal hairpin and implementing promotor with low affinity to RNA polymerase results in stable low-speed replication with medium -range copy number of the plasmid of the invention in E. coli host cells.
In the production of proteins by fermentation technology, it is necessary to have a stable expression system and to maintain a stable occurrence of plasmid after the reduction of antibiotic-induced selection pressure. The loss of the plasmid means loss of the adaptive functions carried by the plasmid and, on the other hand, uncontrolled replication of the plasmid can be lethal for the cell. The regulation of plasmid copy number is driven by regulatory proteins and small RNA molecules that respond to the environmental changes much faster and with lower energy requests than the traditional expression of genetic information. Plasmid of the invention contains in its complete ORI sequence a newly designed P2 promoter and a stabilizing element, which is a sequence identical to the terminal sequence of the replication initiation protein (terminal amino acid sequence of the RepE protein). The RepE protein plays an essential role in initiating replication from the origin, ori2. The RepE protein has two main functions: initiation of replication from the origin, ori2, and autogenous repression of repE transcription. Monomers of RepE represent the active form by binding to ori2 to initiate replication, while dimers act as an autogenous repressor by binding to the operator. Increased expression of the RepE gene switches the initiation of replication to autogenous repression, which leads to a reduction in replication and thus to a reduction in the number of plasmid copies. Thus, the stabilizing element regulates the number of RepE protein molecules so that the replication of the plasmid is not blocked. This occurs through hybridization of the antisense RNA with the complementary region of the gene for the RepE protein. The plasmid of the invention was tested for stability in two host strains, DH10β and BL21 (DE3). In both host strains, it showed a comparable stability to the reference plasmid pET28 (Fig. 1) at a one third of the plasmid size.
Gene encoding ubiquitin:
The ubiquitin (Ub) gene encodes a protein that interacts with most other proteins with a Kdd affinity > 0.3 mM, which obscures the hydrophobic segments of the fusion protein with a direct effect on protein expression and solubility. Originally, ubiquitin was an eukaryotic 8.5 kDa protein with many functions. Its sequence is highly conserved from unicellular organisms to humans. Its presence in the sequence facilitates the production, isolation and identification of the protein from the host production system. Ubiquitin also increases the solubility of the fusion protein produced (Rogov VV, Rozenknop A, Rogova NY, Lohr F, Tikole S, Jaravine V, Gimtert P, Dikic I, Dotsch V. A universal expression tag for structural and functional studies of proteins. Chembiochem. 2012 May 7;13(7):959-63. doi: 10.1002/cbic.201200045. Epub 2012 Mar 20. PMID: 22434781).
In the plasmid of the invention, the gene encoding ubiquitin is optimized for codon usage in E. colt and preferably has a DNA sequence SEQ ID NO: 3. This gene sequence corresponds to the amino acid sequence SEQ ID NO: 4.
SEQ ID NO: 3:
5 ' -ATGCAGATCTTCGTTAAAACCCTGACCGGTAAAACCATCACCCTGGAAGTTGAACCGTCTGACACCATCGAA AACGTTAAAGCGAAAATCCAGGACAAAGAAGGTATCCCGCCGGACCAGCAGGAACTGATCTTCGCGGGTAAACAG CTGGAAGACGGTCGTACCCTGTCTGACTACAACATCCAGAAAGAATCTACCCTGCACCTGGTTCTGCAGCTCGAG TCTGCC-3 ' atg cag atc ttc gtt aaa acc ctg acc ggt aaa acc atc acc ctg gaa gtt gaa ccg tct
M Q I F V K T L T G K T I T L E V E P S gac acc atc gaa aac gtt aaa gcg aaa atc cag gac aaa gaa ggt atc ccg ccg gac cag
D T I E N V K A K I Q D K E G I P P D Q cag gaa ctg atc ttc gcg ggt aaa cag ctg gaa gac ggt cgt acc ctg tct gac tac aac
Q E L I F A G K Q L E D G R T L S D Y N atc cag aaa gaa tct acc ctg cac ctg gtt ctg cag etc gag tct gcc ( SEQ ID NO : 3 )
I Q K E S T L H L V L Q L E S A ( SEQ I D NO : 4 )
Multiple cloning site (MCS)
The DNA sequence for a recombinantly produced protein is inserted into the multiple cloning site (MCS) of the expression vector. The multiple cloning site or polylinker forms a short DNA segment (SEQ ID NO: 5) that contains a series of 8 restriction enzyme sites (RE) which are sites for Kpnl, Ncol, BamHI, PstI, SacI, Hindlll, Notl, Sall.
SEQ ID NO: 5:
5 ' -GGTACCATGGTTGGATCCGCTGCAGGAGCTCAAGCTTGCGGCCGCAGTCGAC-3 '
Kpnl Neol BamHI PstI SacI Hindlll Notl Sal l
GGTACCATGGTTGGATCCGCTGCAGGAGCTCAAGCTTGCGGCCGCAGTCGAC ( SEQ I D NO : 5 )
Affinity tags:
The expression vector of the invention contains nucleotide sequences encoding peptide tag enabling detection and purification of recombinant protein product out of the crude biological source by an affinity technique.
The plasmid of the invention preferably contains two versions of polyhistidine tags in its sequence, a 14xHisTag and a 6xHisTag.
A peptide tag containing 14 histidines (14xHisTag) and additional amino acids (G, S and T) forming loops in its secondary structure is attached on the N-terminus of a recombinantly produced protein. Due to its structure, the 14xHisTag elicits higher affinity to divalent cations and therefore may improve the purification process by metallochelating methods. Proteins expressed from the vector plasmid of the invention have the ability to cleave the 14xHisTag after purification due to the presence of TEV cleavage site in between of a tag sequence and the protein of interest.
The 14xHisTag DNA sequence is preferably SEQ ID NO: 6, and the corresponding amino acid sequence is SEQ ID NO: 7.
SEQ ID NO: 6:
5 ' -CACCACCACCACTCTGGTCACCACCACACGGGTCACCACCACCACTCTGGTTCTCACCACCAC- 3 ' cac cac cac cac tct ggt cac cac cac acg ggt cac cac cac cac tct ggt tct cac H H H H S G H H H T G H H H H S G S H cac cac ( SEQ ID NO : 6 ) H H ( SEQ ID NO : 7 )
In this preferred embodiment, the expression vector of the invention also contains a conventional 6xHisTag sequence behind the multiple cloning site (MCS), which can be used as a C-terminal peptide tag for the metallochelation carrier. The presence of 6xHisTag in a protein sequence may improve detection of a protein by commercially available specific anti-HisTag antibody. When cloning, the gene of interest (GOI, gene encoding the recombinant protein to be produced) must be in the same open reading frame without a stop codon.
The sequence encoding 6xHisTag is SEQ ID NO: 8, and the corresponding amino acid sequence is SEQ ID NO: 9.
SEQ ID NO: 8:
5 ' -CAC CAC CAC CAC CAC CAC -3 ' cac cac cac cac cac cac ( SEQ ID NO : 8 )
H H H H H H ( SEQ ID NO : 9 )
TEV site:
TEV site is a short amino acid sequence enabling the cleavage of ubiquitin sequence and 14xHisTag from the recombinantly produced protein by a TEV protease.
TEV cleavage site is in the plasmid of the invention encoded by a DNA sequence SEQ ID NO: 10, and the corresponding amino acid sequence is SEQ ID NO: 11.
SEQIDNO: 10:
5 ' -GAAAACCTGTACTTCCAGTCT-3 ' gaa aac ctg tac ttc cag tct (SEQ ID NO: 10)
E N L Y F Q S (SEQ ID NO: 11)
Gene for kanamycin resistance:
The preferred selection antibiotic is kanamycin, and the preferred selection antibiotic resistance gene is a codon optimised kanamycin resistance gene having the sequence SEQ ID NO: 12. The kanamycin resistance gene KmR, which is used in a number of E. coli expression plasmids, including pET plasmids, was originally isolated from transposable element Tn903 in E. coli. This gene encodes an aminoglycoside 3 '-phosphotransferase (kanamycin kinase; neomycin-kanamycin phosphotransferase). Aminoglycoside phosphotransferases inactivate aminoglycoside antibiotics through phosphorylation. The original gene had a CAI (Codon adaptation index) of 0.39 and was improved to 0.95 after codon usage optimization.
The kanamycin resistance gene sequence is preferably SEQ ID NO: 12, its complementary sequence is SEQ ID NO: 13, and the corresponding amino acid sequence is SEQ ID NO: 14.
SEQ ID NO: 12:
5 ' -TTAGAAAAACTCATCCAGCATCAGGTGAAATTGCAGCTTATTCATATCCGGGTTGTCGATGCCATATTTTTG AAACAGACGCTTTTGCAGGCTCGGGCTGAACTCGCCCAGGCAGTTCCACAGAATCGCCAGATCTTGGTAACGGTC CGCGATGCCAACACGACCCACATCAATGCAGCCGATCAGTTTACCCTCGTCAAAGATCAGGTTATCCAGGCTGAA GTCGCCGTGGGTAACCACGCTATCCGGGCTAAACGGCAGCAGTTTGTGCATTTCCTTCCAAACCTGCTCCACCGG CCAGCCGTTACGTTCATCGTCGAAATCGCTCGCGTCAACCAGACCGTTGTTCATACGGCTTTGCGCCTGCGCCAG ACGAAAAACACGGTCGCTGTTGAACGGGCAGTTGCACACCGGAATGCTGTGCAGACGACGCAGAAAAACCGCCAG CGCATCCACGATGTTCTCGCCGCTGTCCGGGTATTCCTCCAGCACTTGGAACGCGGTTTTACCCGGAATCGCGGT GGTCAGCAGCCACGCATCATCCGGGGTACGAATAAAGTGCTTGATGGTCGGCAGCGGCATGAACTCGGTCAGCCA GTTCAGACGAACCATCTCATCGGTCACGTCGTTCGCCACGCTGCCTTTACCGTGCTTCAGGAACAGCTCCGGCGC ATCCGGCTTGCCATACAGACGGTAGATGGTCGCGCCGCTCTGACCCACGTTGTCACGCGCCCACTTATAACCATA CAGATCCGCATCCATGTTGCTATTCAGACGCGGACGGCTACAGCTGGTCTCACGTTGAATGTGCAT-3 ' atg cac att caa cgt gag acc agc tgt agc cgt ccg cgt ctg aat agc aac atg gat gcg
M H I Q R E T S C S R P R L N S N M D A gat ctg tat ggt tat aag tgg gcg cgt gac aac gtg ggt cag agc ggc gcg acc atc tac
D L Y G Y K W A R D N V G Q S G A T I Y cgt ctg tat ggc aag ccg gat gcg ccg gag ctg ttc ctg aag cac ggt aaa ggc agc gtg
R L Y G K P D A P E L F L K H G K G S V
gcg aac gac gtg acc gat gag atg gtt cgt ctg aac tgg ctg acc gag ttc atg ccg ctg
A N D V T D E M V R L N W L T E F M P L ccg acc atc aag cac ttt att cgt acc ccg gat gat gcg tgg ctg ctg acc acc gcg att
P T I K H F I R T P D D A W L L T T A I ccg ggt aaa acc gcg ttc caa gtg ctg gag gaa tac ccg gac agc ggc gag aac atc gtg
P G K T A F Q V L E E Y P D S G E N I V gat gcg ctg gcg gtt ttt ctg cgt cgt ctg cac agc att ccg gtg tgc aac tgc ccg ttc
D A L A V F L R R L H S I P V C N C P F aac agc gac cgt gtt ttt cgt ctg gcg cag gcg caa agc cgt atg aac aac ggt ctg gtt N S D R V F R L A Q A Q S R M N N G L V gac gcg agc gat ttc gac gat gaa cgt aac ggc tgg ccg gtg gag cag gtt tgg aag gaa
D A S D F D D E R N G W P V E Q V W K E atg cac aaa ctg ctg ccg ttt agc ccg gat agc gtg gtt acc cac ggc gac ttc agc ctg
M H K L L P F S P D S V V T H G D F S L gat aac ctg atc ttt gac gag ggt aaa ctg atc ggc tgc att gat gtg ggt cgt gtt ggc
D N L I F D E G K L I G C I D V G R V G atc gcg gac cgt tac caa gat ctg gcg att ctg tgg aac tgc ctg ggc gag ttc agc ccg
I A D R Y Q D L A I L W N C L G E F S P agc ctg caa aag cgt ctg ttt caa aaa tat ggc atc gac aac ccg gat atg aat aag ctg S L Q K R L F Q K Y G I D N P D M N K L caa ttt cac ctg atg ctg gat gag ttt ttc taa (SEQ ID NO: 13)
Q F H L M L D E F F - (SEQ ID NO: 14)
Brief description of drawings
Figtire 1. The plasmid of the invention stability in E. coli BL21 (DE3) cells - a comparative study of plasmid retention under non-selective conditions for four subsequent passages. Bars indicate optical density after 24 h of cultivation with and without the addition of antibiotics. On the left side, results for cells transformed with the plasmid having SEQ ID NO: 1 are depicted. On the right side, results of control cells transformed with pET28 are depicted.
Figure 2. The plasmid map comprises the DNA sequence of SEQ ID NO: 2 encoding ORI gene, the ubiquitin leader protein of SEQ ID NO: 3, 14xHisTag sequence for affinity purification (SEQ ID NO: 6) and optimized gene for kanamycin resistance (SEQ ID NO: 12).
The DNA sequence for the gene of interest (GOI) can be inserted into the multiple cloning site (MCS - (SEQ ID NO: 5) of the expression vector plasmid of the invention. A multiple cloning site or polylinker forms a short segment of DNA that contains a series (generally up to 8 REs). Restriction enzyme (RE) sites - are Kpnl, Ncol, BamHI, PstI, Sad, Hindlll, Notl, Sall.
Examples
Assembling of the plasmid
A plasmid according to the invention having SEQ ID NO: 1 (plasmid pUbExlOO) was created by design de novo synthetically. The linear plasmid was circularized by ligation and, after transformation into the target host cells, was propagated in them and subsequently isolated.
Cloning - General procedure
Inserting a coding sequence of a protein to be produced (gene of interest) into the plasmid of the invention
The DNA sequence for a recombinantly produced protein (gene of interest) is inserted into the multiple cloning site (MCS) of the expression vector plasmid having the sequence SEQ ID NO: 1 (plasmid pUbExlOO). The multiple cloning site is formed by a short DNA segment that contains a series of short DNA sequences recognised by restriction enzymes in following order Kpnl, Ncol, BamHI, PstI, Sad, Hindlll, Notl, Sall. A cleavage site may be included between the leader protein Ubq with 14xHis-Tag peptide for IMAC affinity purification (Immobilized Metal Chelate Affinity Chromatography) and the protein of interest to cleave off the leader peptide and purify recombinant protein. The gene of interest may be inserted in any of the cleavage sites in the MCS. All genes were codon optimised and shared common restriction sites at 5’- and 3’- ends of coding sequences. The incorporation procedure, as well as protein expression experiments, were done under the same conditions.
Gene for the recombinantly produced protein (gene of interest) was processed by restriction enzymes Ncol/Notl in CutSmart restriction Buffer (New England Biolabs, USA) for 1 hour at 37°C. The restriction mixture was separated by DNA electrophoresis. The DNA fragment of appropriate size was excised and isolated by isolation kit NucleoSpin Gel and PCR Clean-up (Macherey-Nagel). The DNA concentration was determined spectrophotometrically. The vector was linearised by the same set of restriction enzymes. The incorporation of the gene of interest was achieved via DNA ligation. Ten ng of the insert was incubated with 100 ng of linearised vector plasmid of the invention in the presence of T4 DNA ligase for 12 hours at 16°C. The reaction product was used for heat-shock transformation of E. coli DHIOp chemo-competent cells. Cultivation on agar plates with kanamycin selection was used to select cells carrying the plasmid. The presence of the gene of interest in the plasmid was confirmed by DNA sequencing.
Plasmid transformation
Vector plasmid of the invention containing the gene of interest coding sequence was used to transform production strain E. coli BL21 (DE3). 50 ng of plasmid DNA was added to chemo -competent cells, which were transformed by heat shock (42° C/60 sec.). Positive clones were selected using seeding on LB agar plates with kanamycin. Fifteen colonies from the Petri dish were transferred to a liquid LB medium with kanamycin (100 pg/ml) and glucose (1 %).
Cultivating the prokaryotic host cells
The inoculum was cultivated at 37°C for 16 hours under constant agitation at 220 rpm. 1 ml of cell culture was used to inoculate 100 ml of sterile autoinduction media (composition for IL: 10 g Tryptone; 5 g Yeast extract; 3.3 g ammonium sulphate; 50 mM phosphate buffer pH 6.7; 0.5 g glucose; 2 g lactose). The cultivation was kept for 48 hours at 22°C under constant agitation at 220 rpm.
Cell harvest, disintegration and protein purification
The cultivation was terminated by cooling down the cell suspension to 4°C. The cells were harvested by centrifugation (6000 g/4°C/10 min.). The bacterial pellet was resuspended in the appropriate buffer (e.g. 100 mM Tris-HCl pH 8, 300 mM NaCl, 0.1 % Tween 20 (v/v), 10 mM Imidazole) and subsequently disintegrated by high pressure homogenisation. Homogenised cell suspension was centrifuged for one hour at 4°C and 14000 g, fdtrated through 0.22 pm fdter and loaded in the same buffer to IMAC column. Non-specifically bound proteins were washed by increasing the concentration of imidazole to 125 mM. The recombinantly produced protein was eluted by 250 mM imidazole in the same buffer.
Optional leader protein cleavage
The TEV protease processing can be used for leader protein removal. Purified protein was dialysed against the same buffer as used for purification except the imidazole (e.g. 100 mM Tris-HCl pH 8, 300 mM NaCl, 0.1 % Tween 20 (v/v)). Dialysed protein was incubated with TEV protease in the 1:30 ratio (w/w) for 16 hours at 4°C. Cleaved mixture was loaded on the IMAC column and the unbound protein (where the leader protein including his-tag was cleaved) is collected.
Stability of the plasmid of the invention
Determination of the plasmid of the invention stability.
The stability of the plasmid having the sequence SEQ ID NO: 1 in E. coli genome was verified. Two commonly used bacterial strains, DH10β and BL21 (DE3), were used in a series of cultivation experiments as described. Freshly transformed bacteria, either with the plasmid of the invention or pET28, with an incorporated gene for lysostaphin protein, were cultivated on agar plates at 37°C with appropriate antibiotics for 16 hours. A single colony of each strain was picked and used to inoculate
liquid broth without antibiotics and cultivated for 24 hours at 37°C under agitation. The optical density was measured for each culture, and 100 pl was used to inoculate freshly prepared liquid broth without antibiotics so that the starting optical density was 0.5 MacFarland. At the same time, the test for plasmid presence was performed as follows. 10 pl of liquid culture was used to inoculate 10 ml of liquid broth with antibiotics, and 20 pl was seeded onto the agar plate with antibiotics. The optical density and colony counting were performed after 16 h of cultivation at 37°C. Five subsequent passages were done, and the colony counting, and optical density measurements were performed for each of them.
The result confirmed the stable maintenance of the plasmid according to the invention even without antibiotic selection for at least five passages, with an insignificant effect on the growth ability when antibiotics are added. No significant difference was found compared to another commonly used expression plasmid pET28. The newly designed ORI sequence of the remainder of the pUbExlOO plasmid proved suitable for long-term cultivation (at least five days) without antibiotics. The novel plasmid of the invention is cost-effective and can reduce the use of antibiotics in large-scale protein production.
Determination of the copy number of plasmid of the invention per cell
A combination of plasmid DNA isolation methods, cell number counting and quantitative polymerase chain reaction (qPCR) was used to determine the copy number of plasmid DNA in the low copy, medium copy or high copy plasmid range. Copy number was determined against standard high copy (pUC57) and low copy (pET28) plasmids. All tests described below were performed in technically and biologically independent triplicates.
The growth rate of A. coll DHIOp chemically competent cells transformed with plasmids pUC57, pET28 or the plasmid of the invention, in which marker gene for lysostaphin protein was cloned was determined. Heat-shock transformation in KCM buffer and subsequent cultivation on microbiological LB agar with the addition of suitable antibiotics - kanamycin for pET28 and of the invention, and ampicillin for pUC57 were used for the transformation. Cultivation was performed at 37°C for 16 hours. Three colonies were randomly selected from each plate and inoculated into a liquid LB medium with a suitable antibiotic (ampicillin or kanamycin). Cultivation was continued with stirring (180 rpm) at 37°C for 16 hours. 150 ml of the grown liquid cultures were removed and transferred to 5 ml of LB medium with the addition of antibiotics. Cultures were grown at 37CC with constant agitation until an optical density of 1.2 MacFarland was reached. The cultures were rapidly cooled on ice and serially diluted in LB medium with antibiotics in the concentration range of 101 - 109 by transferring 100 pl of bacterial suspension to 900 pl of LB medium. Five pl of each dilution was plated on agar plates with the appropriate antibiotic.
At the same time, plasmid DNA isolations for all described cultures (9 cultures) were done using EZNA Plasmid Mini Kit 1 (Omega Bio-Tek). The isolation procedure was performed according to the
manufacturer's instructions. 4 ml of cell suspension from each culture (9 plasmid DNA isolations) were used as input material. The isolated plasmid DNA was eluted with 60 pl of elution buffer, and the plasmid DNA concentration was determined spectrophotometrically. The isolated plasmid DNA was serially diluted for each of the samples in demineralized water in a quality suitable for PCR at a decimal dilution from 101 - 1012. The qPCR amplification Ct value was determined for all these samples.
Gene for Lysostaphin SEQ ID NO: 15:
ATGGCGCACGAGCACAGCGCGCAGTGGCTGAACAACTACAAGAAGGGTTATGGTTATGGTCCGTATCCGCTGGGT ATCAATGGTGGTATGCACTACGGCGTGGACTTCTTTATGAACATCGGTACCCCGGTGAAGGCGATCAGCAGCGGC AAAATTGTTGAGGCGGGTTGGAGCAACTATGGTGGCGGTAACCAGATCGGCCTGATTGAAAACGACGGTGTGCAC CGTCAATGGTACATGCACCTGAGCAAGTATAACGTGAAAGTTGGCGATTACGTTAAGGCGGGTCAGATCATTGGC TGGAGCGGTAGCACCGGTTATAGCACCGCGCCGCACCTGCACTTCCAGCGTATGGTGAACAGCTTTAGCAACAGC ACCGCGCAAGACCCGATGCCGTTCCTGAAGAGCGCGGGTTATGGTAAAGCGGGCGGATCCGTTACCCCGACCCCG AACACCGGCTGGAAGACCAACAAATACGGCACCCTGTATAAAAGCGAGAGCGCGAGCTTCACCCCGAACACCGAT ATCATTACCCGTACCACCGGCCCGTTTCGTAGCATGCCGCAGAGCGGCGTGCTGAAGGCGGGTCAAACCATCCAC TATGACGAAGTTATGAAACAGGATGGTCACGTGTGGGTTGGTTACACCGGCAACAGCGGTCAACGTATTTATCTG CCGGTTCGCACCTGGAATAAAAGCACCAACACCCTGGGCGTTCTGTGGGGTACCATCAAATAA
The primers used for amplification:
Forward primer (+) SEQ ID NO: 16: 5 ' - GTATAACGTGAAAGTTGGCG - 3 '
Reverse primer (-) SEQ ID NO: 17: 5 ' - TCTCGCTTTTATACAGGGTG - 3 '
Both primers define a specific binding region of 248 base pairs in the gene encoding the lysostaphin protein. The reaction temperature profile was composed of an initial denaturation of 95°C for 10 min, 45 cycles alternating 95°C for 10 s, 60°C for 20 s and 72°C for 20 s. After completion of the amplification reaction, the melting point of the PCR products was analysed with an increment of 0.5 °C in the range of 60-95 °C for 30 minutes. Samples were plated in duplicate for all DNA dilutions, for a total of 9 x 240 samples. PCR assay and melting point analysis were performed by detecting a change in fluorescence in the reaction mixture containing SybrGreen intercalation dye.
Determination of the number of cells used for plasmid DNA isolation.
Based on the agreement of the turbidity measurement value on the MacFarland scale (1.3) and evaluation of colony forming units count on solid agar, the cell concentration per ml was determined for each culture, taking into account the average value from the number of replicates: for pUC57 = 106 CFU/ml, pET28 = 106 CFU/ml and plasmid of the invention = 105 CFU/ml.
Determination of relative copy number of plasmid DNA
Plasmid DNA copy number was used based on pET28 low copy (~15 - 20) and pUC57 high copy (-500- 700) standards in cases where at all dilutions the melting point analysis corresponded to a single PCR product of 82.5°C, which corresponds to the expected product.
Based on the Ct value (cycle of threshold) for each isolate relative to the number of cells used for plasmid DNA isolation (CFU). Based on the reported values of reference plasmids pUC57 and pET28, the number of plasmids per cell for pUbExlOO was calculated as the range of 46. 1 - 65.8 +/- 10% copies.
Production of Apxla using the plasmid of the invention
Apxla is related to RTX toxins (APXI - APXIV) of Actinobacilus pleuropneumonia. APXla is a strongly cytotoxic and haemolytic pore-forming factor. Together with capsular polysaccharides and mural lipo-polysaccharides, Apxla is an essential factors of virulence and thus responsible for fibrinhaemorrhagic pleuropneumonia in swine. The DNA sequence coding for Apxla C -terminal domain sequence with E. colt optimized codon usage distribution was synthesized and inserted into the expression vector via restriction enzyme digestion and subsequent ligation. The produced protein is of 27 kDa apparent molecular weight.
Plasmid of the invention with an inserted Apxla gene (SEQ ID NO: 18):
CTCGAGTCTGCCCACCACCACCACTCTGGTCACCACCACACGGGTCACCACCACCACTCTGGTTCTCACCACCAC TCTGGTGCCGAAAACCTGTACTTCCAGTCTGGTTCTGGTACCATGGGATCCTCTGTTGAAGAAATTATCGGTAGT AATCGTAAAGACAAATTCTTTGGTAGTCGCTTTACCGATATTTTCCATGGTGCGAAAGGCGATGATGAAATCTAC GGTAATGACGGCCACGATATCTTATACGGAGACGACGGTAATGATGTAATCCATGGCGGTGACGGTAACGACCAT CTTGTTGGTGGTAACGGAAACGACCGATTAATCGGCGGAAAAGGTAATAATTTCCTTAATGGCGGTGATGGTGAC GATGAGTTGCAGGTCTTTGAGGGTCAATACAACGTATTATTAGGTGGTGCGGGTAATGACATTCTGTATGGCAGC GATGGTACTAACTTATTTGACGGTGGTGTAGGCAATGACAAAATCTACGGTGGTTTAGGTAAGGATATTTATCGC TACAGTAAGGAGTACGGTCGTCATATCATTATTGAGAAAGGCGGTGATGATGATACGTTATTGTTATCGGATCTT
AGTTTTAAAGATGTAGGATTTATCAGAATCGGTGATGATCTTCTTGTGAATAAAAGAATCGGAGGAACACTGTAT TACCATGAAGATTACAATGGGAATGCGCTCACGATTAAAGATTGGTTCAAGGAAGGTAAGCTTTCTAGATAATGA
GCGGCCGCAGTCGACCACCACCACCACCACCACTGAGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTG GCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTG AAAGGAGGAACTATATCCGGATGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTC ATGAATTAATTCTTAGAAAAACTCATCCAGCATCAGGTGAAATTGCAGCTTATTCATATCCGGGTTGTCGATGCC ATATTTTTGAAACAGACGCTTTTGCAGGCTCGGGCTGAACTCGCCCAGGCAGTTCCACAGAATCGCCAGATCTTG GTAACGGTCCGCGATGCCAACACGACCCACATCAATGCAGCCGATCAGTTTACCCTCGTCAAAGATCAGGTTATC CAGGCTGAAGTCGCCGTGGGTAACCACGCTATCCGGGCTAAACGGCAGCAGTTTGTGCATTTCCTTCCAAACCTG CTCCACCGGCCAGCCGTTACGTTCATCGTCGAAATCGCTCGCGTCAACCAGACCGTTGTTCATACGGCTTTGCGC CTGCGCCAGACGAAAAACACGGTCGCTGTTGAACGGGCAGTTGCACACCGGAATGCTGTGCAGACGACGCAGAAA AACCGCCAGCGCATCCACGATGTTCTCGCCGCTGTCCGGGTATTCCTCCAGCACTTGGAACGCGGTTTTACCCGG AATCGCGGTGGTCAGCAGCCACGCATCATCCGGGGTACGAATAAAGTGCTTGATGGTCGGCAGCGGCATGAACTC GGTCAGCCAGTTCAGACGAACCATCTCATCGGTCACGTCGTTCGCCACGCTGCCTTTACCGTGCTTCAGGAACAG CTCCGGCGCATCCGGCTTGCCATACAGACGGTAGATGGTCGCGCCGCTCTGACCCACGTTGTCACGCGCCCACTT ATAACCATACAGATCCGCATCCATGTTGCTATTCAGACGCGGACGGCTACAGCTGGTCTCACGTTGAATGTGCAT AACAC C C CT T GT AT TACT GT T TAT GT AAGCAGACAGT T T TAT T GT T CAT GAC CAAAAT C C CT T AAC GT GAGT T T T CGTTCCACTGAGCGTCAGACCCCGTAGAAAAGAGCGCATGCCGTGAGACTGCGGCGGGCGTTACCGGCTCACAAA
TAACGGGATACGCAGGCAGTGCTCAAATCAGGAAGGACCGGAAAAAGGATGCGGCGTAGCCGTTTTTCCATAGGC TCCGCCCCCCTGACAAGCATCACGAAATCTGACGCTCAAATCAGTGGCGGCGAAACCCGACAGGACTATAAAGAT CCCAGGCGTTTCCCCCTGGTAGCTCCCTCGTGCGCTCTCCTGTTCCTGCCTTTCGGTTTACCGGTGTCATTCCGC TGTTATGGCCGCGTTTGTCTCATTCCACGCCTGACACTCAGTTCCGGGTAGGCAGTTCGCTCCAAGCTGGACTGT ATGCACGAACCCCCCGTTCAGTCCGACTACCACGCCCGTTCCGGTAACTATCAACTTGAGTCCAACCCGGAAAGA CACGACAAATCGCCAGTGGCGGTAGCCATTGGTAACTGAGATGTGCGAGAGATTTATCTGGAGTTCTTGAAGTGG GGGCCTGAGTGCGGCTACACTGGAAGGACAGTTTAGGTGACTCGTCTCGCACAAGACAGTTACCACGGTTCATAA ACTTGCCGGTTTAATGAACCTTCGAAAAACCACCTTGCCGGGTGGTTTTTTCTTTTCAAAGAAGATACGCGTTTA CTCTTCGCCTCGTCAATGAAGGGTCGCATTAATTCGATCCCGCGAAATTAATACGACTCACTATAGGGGAATTGT GAGCGGATAACAATTCCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACATATGCAGATCTTCGT TAAAACCCTGACCGGTAAAACCATCACCCTGGAAGTTGAACCGTCTGACACCATCGAAAACGTTAAAGCGAAAAT CCAGGACAAAGAAGGTATCCCGCCGGACCAGCAGGAACTGATCTTCGCGGGTAAACAGCTGGAAGACGGTCGTAC CCTGTCTGACTACAACATCCAGAAAGAATCTACCCTGCACCTGGTTCTGCAG
Gene for recombinantly produced protein Apxla incorporation into the plasmid of the invention
Gene encoding Apxla was processed by restriction enzymes Ndel/Notl in CutSmart restriction Buffer (New England Biolabs, USA) for 1 hour at 37°C. The restriction mixture was separated using DNA electrophoresis. The DNA fragment of appropriate size was excised and isolated by isolation kit NucleoSpin Gel and PCR Clean-up (Macherey-Nagel). The DNA concentration was determined spectrophotometrically. The vector was linearised by the same procedure. The incorporation of the gene of interest was achieved via DNA ligation. 10 ng of the linearised insert was incubated with 100 ng of
linearised vector plasmid of the invention in the presence of T4 DNA ligase for 12 hours at 16°C. The reaction product was used for heat-shock transformation of E. coli DHIOp chemo-competent cells. Cultivation on agar plates with kanamycin selection was used to select cells carrying the plasmid. The presence of the gene of interest in the vector plasmid of the invention was confirmed by DNA sequencing.
Protein expression experiments
Vector plasmid of the invention containing the gene of interest coding sequence was used to transform production strain E. coli BL21 (DE3). 50 ng of plasmid DNA was added to chemo -competent cells, which were transformed by heat shock (42°C/60 sec.) in the water bath. Positive clones were selected using seeding on LB agar plates with kanamycin. Fifteen colonies from the Petry dish were transferred to a liquid LB medium with kanamycin (100 pg/ml) and glucose (1%). The inoculum was cultivated at 37°C for 12 hours under constant agitation at 220 rpm. 1 ml of cell culture was used to inoculate 100 ml of sterile autoinduction media (composition for IL: 10 g Tryptone; 5 g Yeast extract; 3.3 g ammonium sulphate; 50 mM phosphate buffer pH 6.7; 0.5 g glucose; 2 g lactose). The cultivation was kept for 48 hours at 22°C under constant agitation at 220 rpm. The protein production was verified by protein SDS- PAGE electrophoresis and subsequent Coomasie Brilliant blue staining of whole cell lysates. For three proteins, the production rate was analysed in more detail.
Comparison of expression of the protein Apxla.
The production of Apxla protein was compared with the commonly used laboratory expression plasmid pET. The result shows a comparison of the measured pellet optical density (McFarland 0.5 = 1.5 x 108 CFU/ml) between the new plasmid of the invention and pET28. The difference is most pronounced in the first 24 hours, when plasmid of the invention-Apxla has a density of only 5 McFarland and pET28-Apxla has a density of 21 McFarland. After 48 hours, the density of pET28-Apxla increased to 37 McFarland and pUbExlOO-Apxla to density 33 McFarland. Although the resulting pellet of pUbExlOO-Apxla had a lower yield (12.25 g/L) than pET28-Apxla (15.25 g/L), yet the final protein production was 1.37x greater with the new plasmid pUbExlOO-Apxla.
Reducing the biomass required to produce a specific amount of protein significantly reduces the cost of the downstream process and subsequent protein purification steps. Plasmid pUbExlOO improves the subsequent purification process and makes its production much more efficient.
Production of Bst 5.9 using the plasmid of the invention.
Bst 5.9 polymerase is a chimeric enzyme consisting of the main scaffold of Bst polymerase from Thermus aquations Taq polymerase. A short loop from Bst polymerase from Geobacillus stearothermophilus was introduced. Bst 5.9 polymerase is used in the isothermal amplification reaction. The coding sequence with E. coli optimized codon usage distribution was synthesized and inserted into the expression vector via restriction enzyme digestion and subsequent ligation. The produced protein is of 72 kDa apparent molecular weight.
Plasmid of the invention with an inserted Bst 5.9 gene (SEQ ID NO: 19)
CTCGAGTCTGCCCACCACCACCACTCTGGTCACCACCACACGGGTCACCACCACCACTCTGGTTCTCACCACCAC TCTGGTGCCGAAAACCTGTACTTCCAGTCTGGTTCTGGTACCATGGTTGGATCCCTGCTGCACGAGTTTGGTCTG CTGGAAAGCCCGAAAGCGCTGGAAGAGGCGCCGTGGCCGCCGCCGGAGGGTGCGTTCGTGGGCTTCGTTCTGAGC CGTAAAGAGCCGATGTGGGCGGATCTGCTGGCGCTGGCGGCGGCGCGTGGTGGCCGTGTGCACCGTGCGCCGGAA CCGTACAAAGCGCTGCGTGACCTGAAAGAAGCGCGTGGTCTGCTGGCGAAGGACCTGAGCGTGCTGGCGCTGCGT GAGGGCCTGGGTCTGCCGCCGGGCGATGACCCGATGCTGCTGGCGTACCTGCTGGACCCGAGCAACACCACCCCG GAAGGTGTTGCGCGTCGTTACGGCGGTGAATGGACCGAAGAGGCGGGCGAACGTGCGGCGCTGAGCGAACGTCTG TTCGCGAACCTGTGGGGTCGTCTGGAGGGTGAAGAGCGTCTGCTGTGGCTGTACCGTGAGGTTGAACGTCCGCTG AGCGCGGTTCTGGCGCACATGGAGGCGACCGGTGTTCGTCTGGATGTTGCGTATCTGCGTGCGCTGAGCCTGGAA GTGGCGGAAGAGATCGCGCGTCTGGAAGCGGAAGTGTTCCGTCTGGCGGGTCACCCGTTTAACCTGAACAGCCGT GATCAACTGGAACGTGTTCTGTTCGATGAACTGGGCCTGCCGGCGATTGGCAAAACCGAAAAAACCGGCAAGCGT AGCACCAGCGCGGCGGTGCTGGAAGCGCTGCGTGAAGCGCACCCGATTGTTGAAAAAATTCTGCAATACCGTGAG CTGACCAAGCTGAAAAGCACCTACATTGAGGGTCTGCTGAAGGTTGTGCGTCCGGATACCAAGAAAGTTCACACC CGTTTTAACCAGACCGCGACCGCGACCGGTCGTCTGAGCAGCAGCGACCCGAACCTGCAAAACATCCCGGTGCGT ACCCCGCTGGGTCAGCGTATTCGTCGTGCGTTTATCGCGggtGAGGGTTGGCTGCTGGTTGCGCTGGATTACAGC CAGATCGAACTGCGTGTTCTGGCGCACCTGAGCGGTGACGAGAACCTGATTCGTGTGTTCCAGGAAGGCCGTGAC ATCCACACCGAGACCGCGAGCTGGATGTTTGGTGTGCCGCGTGAGGCGGTTGACCCGCTGATGCGTCGTGCGGCG AAGACCATCAACTTTGGCGTTCTGTACGGTATGAGCGCGCACCGTCTGAGCCAAGAACTGGCGATCCCGTATGAG GAAGCGCAAGCGTTTATCGAGCGTTATTTCCAGAGCTTCCCGAAAGTTCGTGCGTGGATCGAGAAGACCCTGGAA GAGGGCCGTCGTCGTGGTTACGTGGAGACCCTGTTTGGTCGTCGTCGTTACGTGCCGGACCTGGAGGCGCGTGTT AAGAGCGTGCGTGAGGCGGCGGAACGTATGGCGTTTAACATGCCGGTTCAAGGCACCGCGGCGGATCTGATGAAG agcGCGATGGTGAAGCTGTTCCCGCGTCTGGAAGAAATGGGTGCGCGTATGCTGCTGCAAGTTCACGATGAGCTG GTTCTGGAAGCGCCGAAGGAACGTGCGGAAGCGGTTGCGCGTCTGGCGAAGGAAGTGATGGAAGGCGTGTATCCG
CTGGCGGTTCCGCTGGAAGTTGAAGTTGGTATTGGCGAGGATTGGCTGAGCGCGAAAGAGGTCGACTAAGCGGCC
GCAGTCGACCACCACCACCACCACCACTGAGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCT GCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGA GGAACTATATCCGGATGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAAT TAATTCTTAGAAAAACTCATCCAGCATCAGGTGAAATTGCAGCTTATTCATATCCGGGTTGTCGATGCCATATTT TTGAAACAGACGCTTTTGCAGGCTCGGGCTGAACTCGCCCAGGCAGTTCCACAGAATCGCCAGATCTTGGTAACG GTCCGCGATGCCAACACGACCCACATCAATGCAGCCGATCAGTTTACCCTCGTCAAAGATCAGGTTATCCAGGCT GAAGTCGCCGTGGGTAACCACGCTATCCGGGCTAAACGGCAGCAGTTTGTGCATTTCCTTCCAAACCTGCTCCAC CGGCCAGCCGTTACGTTCATCGTCGAAATCGCTCGCGTCAACCAGACCGTTGTTCATACGGCTTTGCGCCTGCGC CAGACGAAAAACACGGTCGCTGTTGAACGGGCAGTTGCACACCGGAATGCTGTGCAGACGACGCAGAAAAACCGC CAGCGCATCCACGATGTTCTCGCCGCTGTCCGGGTATTCCTCCAGCACTTGGAACGCGGTTTTACCCGGAATCGC GGTGGTCAGCAGCCACGCATCATCCGGGGTACGAATAAAGTGCTTGATGGTCGGCAGCGGCATGAACTCGGTCAG CCAGTTCAGACGAACCATCTCATCGGTCACGTCGTTCGCCACGCTGCCTTTACCGTGCTTCAGGAACAGCTCCGG CGCATCCGGCTTGCCATACAGACGGTAGATGGTCGCGCCGCTCTGACCCACGTTGTCACGCGCCCACTTATAACC ATACAGATCCGCATCCATGTTGCTATTCAGACGCGGACGGCTACAGCTGGTCTCACGTTGAATGTGCATAACACC CCTTGTATTACTGTTTATGTAAGCAGACAGTTTTATTGTTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCC ACTGAGCGTCAGACCCCGTAGAAAAGAGCGCATGCCGTGAGACTGCGGCGGGCGTTACCGGCTCACAAATAACGG GATACGCAGGCAGTGCTCAAATCAGGAAGGACCGGAAAAAGGATGCGGCGTAGCCGTTTTTCCATAGGCTCCGCC CCCCTGACAAGCATCACGAAATCTGACGCTCAAATCAGTGGCGGCGAAACCCGACAGGACTATAAAGATCCCAGG CGTTTCCCCCTGGTAGCTCCCTCGTGCGCTCTCCTGTTCCTGCCTTTCGGTTTACCGGTGTCATTCCGCTGTTAT GGCCGCGTTTGTCTCATTCCACGCCTGACACTCAGTTCCGGGTAGGCAGTTCGCTCCAAGCTGGACTGTATGCAC GAACCCCCCGTTCAGTCCGACTACCACGCCCGTTCCGGTAACTATCAACTTGAGTCCAACCCGGAAAGACACGAC AAATCGCCAGTGGCGGTAGCCATTGGTAACTGAGATGTGCGAGAGATTTATCTGGAGTTCTTGAAGTGGGGGCCT GAGTGCGGCTACACTGGAAGGACAGTTTAGGTGACTCGTCTCGCACAAGACAGTTACCACGGTTCATAAACTTGC CGGTTTAATGAACCTTCGAAAAACCACCTTGCCGGGTGGTTTTTTCTTTTCAAAGAAGATACGCGTTTACTCTTC GCCTCGTCAATGAAGGGTCGCATTAATTCGATCCCGCGAAATTAATACGACTCACTATAGGGGAATTGTGAGCGG ATAACAATTCCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACATATGCAGATCTTCGTTAAAAC CCTGACCGGTAAAACCATCACCCTGGAAGTTGAACCGTCTGACACCATCGAAAACGTTAAAGCGAAAATCCAGGA CAAAGAAGGTATCCCGCCGGACCAGCAGGAACTGATCTTCGCGGGTAAACAGCTGGAAGACGGTCGTACCCTGTC TGACTACAACATCCAGAAAGAATCTACCCTGCACCTGGTTCTGCAG
Gene of interest i corporation into the plasmid of the invention
Gene of interest was processed by restriction enzymes Ndel/Notl in CutSmart restriction Buffer (New England Biolabs, USA) for 1 hour at 37°C. The restriction mixture was separated using DNA electrophoresis. The DNA fragment of appropriate size was excised and isolated by isolation kit NucleoSpin Gel and PCR Clean-up (Macherey-Nagel). The DNA concentration was determined spectrophotometrically. The vector plasmid of the invention was linearised by the same set of restriction enzymes and the same procedure. The incorporation of the gene of interest was achieved via DNA ligation. Ten ng of the linearised insert was incubated with 100 ng of linearised vector plasmid of the
invention in the presence of T4 DNA ligase for 12 hours at 16°C. The reaction product was used for heat-shock transformation of E. coli DH10β chemo-competent cells. Cultivation on agar plates with kanamycin selection was used to select cells carrying the plasmid. The presence of the gene of interest in the vector plasmid of the invention was confirmed by DNA sequencing.
Protein expression experiments
Vector plasmid of the invention containing the gene of interest coding sequence was used to transform production strain E. coli BL21 (DE3). 50 ng of plasmid DNA was added to chemo -competent cells, which were transformed by heat shock (42°C/60 sec.) in the water bath. Positive clones were selected using seeding on LB agar plates with kanamycin. Fifteen colonies from the Petri dish were transferred to a liquid LB medium with kanamycin (100 pg/ml) and glucose (1 %). The inoculum was cultivated at 37°C for 12 hours under constant agitation at 220 rpm. 1 ml of cell culture was used to inoculate 100 ml of sterile autoinduction media (composition for IL: 10 g Tryptone; 5 g Yeast extract; 3.3 g ammonium sulphate; 50 mM phosphate buffer pH 6.7; 0.5 g glucose; 2 g lactose). The cultivation was kept for 48 hours at 22°C under constant agitation at 220 rpm. The protein production was verified by protein SDS- PAGE electrophoresis and subsequent Coomasie Brilliant blue staining of whole cell lysates. For three proteins, the production rate was analysed in more detail.
Comparison of expression of the protein Bst 5.9.
The production of Bst 5.9 protein was compared with the commonly used laboratory expression plasmid pET. The result shows a comparison of the measured pellet optical density (McFarland 0.5 = 1.5 x 108 CFU/ml) between the new plasmid of the invention and pET28. The difference is most pronounced in the first 24 hours, when the plasmid of the invention-Bst5.9 has a density of only 3.5 McFarland and pET28-Bst5.9 has a density of 25 McFarland. After 48 hours, the density of pET28-Bst5.9 increased to 35 McFarland and pUbExlOO-Apxla to 42 McFarland. The resulting pUbExl00-Bst5.9 pellet had a higher yield (14.5 g/L) than pET28-Bst5.9 (8.75 g/L). The final protein production was 3.3-fold higher with the new plasmid of the invention-Apxla. Due to the relatively higher production of protein per unit of biomass, the plasmid of the invention improves the subsequent purification process and makes its production significantly more efficient.
Production of lysostaphin using the plasmid of the invention.
Lysostaphin is a 27 kDa metalloendopeptidase, the antimicrobial lytic enzyme that is produced by Staphylococcus simulans. Lysostaphin is highly active against Staphylococcus aureus strains. The bacteriolytic activity of lysostaphin is due to its ability to hydrolyse the penta-glycine cross bridge of .S' aureus peptidoglycan. The coding sequence with E. coli-optimized codon usage distribution was synthesized and inserted into the expression vector via restriction enzyme digestion and subsequent ligation.
Plasmid of the invention with an inserted Lysostaphine gene (SEQ ID NO: 20):
CTCGAGTCTGCCCACCACCACCACTCTGGTCACCACCACACGGGTCACCACCACCACTCTGGTTCTCACCACCAC TCTGGTGCCGAAAACCTGTACTTCCAGTCTGGTTCTGGTACCATGGCGCACGAGCACAGCGCGCAGTGGCTGAAC AACTACAAGAAGGGTTATGGTTATGGTCCGTATCCGCTGGGTATCAATGGTGGTATGCACTACGGCGTGGACTTC TTTATGAACATCGGTACCCCGGTGAAGGCGATCAGCAGCGGCAAAATTGTTGAGGCGGGTTGGAGCAACTATGGT GGCGGTAACCAGATCGGCCTGATTGAAAACGACGGTGTGCACCGTCAATGGTACATGCACCTGAGCAAGTATAAC GTGAAAGTTGGCGATTACGTTAAGGCGGGTCAGATCATTGGCTGGAGCGGTAGCACCGGTTATAGCACCGCGCCG CACCTGCACTTCCAGCGTATGGTGAACAGCTTTAGCAACAGCACCGCGCAAGACCCGATGCCGTTCCTGAAGAGC GCGGGTTATGGTAAAGCGGGCGGATCCGTTACCCCGACCCCGAACACCGGCTGGAAGACCAACAAATACGGCACC CTGTATAAAAGCGAGAGCGCGAGCTTCACCCCGAACACCGATATCATTACCCGTACCACCGGCCCGTTTCGTAGC ATGCCGCAGAGCGGCGTGCTGAAGGCGGGTCAAACCATCCACTATGACGAAGTTATGAAACAGGATGGTCACGTG TGGGTTGGTTACACCGGCAACAGCGGTCAACGTATTTATCTGCCGGTTCGCACCTGGAATAAAAGCACCAACACC CTGGGCGTTCTGTGGGGTACCATCAAATAAGCGGCCGCAGTCGACCACCACCACCACCACCACTGAGATCCGGCT GCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCC TCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGATGCGGAACCCCTATTTGTTTATTT TTCTAAATACATTCAAATATGTATCCGCTCATGAATTAATTCTTAGAAAAACTCATCCAGCATCAGGTGAAATTG CAGCTTATTCATATCCGGGTTGTCGATGCCATATTTTTGAAACAGACGCTTTTGCAGGCTCGGGCTGAACTCGCC CAGGCAGTTCCACAGAATCGCCAGATCTTGGTAACGGTCCGCGATGCCAACACGACCCACATCAATGCAGCCGAT CAGTTTACCCTCGTCAAAGATCAGGTTATCCAGGCTGAAGTCGCCGTGGGTAACCACGCTATCCGGGCTAAACGG CAGCAGTTTGTGCATTTCCTTCCAAACCTGCTCCACCGGCCAGCCGTTACGTTCATCGTCGAAATCGCTCGCGTC AACCAGACCGTTGTTCATACGGCTTTGCGCCTGCGCCAGACGAAAAACACGGTCGCTGTTGAACGGGCAGTTGCA CACCGGAATGCTGTGCAGACGACGCAGAAAAACCGCCAGCGCATCCACGATGTTCTCGCCGCTGTCCGGGTATTC CTCCAGCACTTGGAACGCGGTTTTACCCGGAATCGCGGTGGTCAGCAGCCACGCATCATCCGGGGTACGAATAAA GTGCTTGATGGTCGGCAGCGGCATGAACTCGGTCAGCCAGTTCAGACGAACCATCTCATCGGTCACGTCGTTCGC CACGCTGCCTTTACCGTGCTTCAGGAACAGCTCCGGCGCATCCGGCTTGCCATACAGACGGTAGATGGTCGCGCC GCTCTGACCCACGTTGTCACGCGCCCACTTATAACCATACAGATCCGCATCCATGTTGCTATTCAGACGCGGACG GCTACAGCTGGTCTCACGTTGAATGTGCATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAGTTTTATTGT TCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGAGCGCATGCCGTG AGACTGCGGCGGGCGTTACCGGCTCACAAATAACGGGATACGCAGGCAGTGCTCAAATCAGGAAGGACCGGAAAA AGGATGCGGCGTAGCCGTTTTTCCATAGGCTCCGCCCCCCTGACAAGCATCACGAAATCTGACGCTCAAATCAGT GGCGGCGAAACCCGACAGGACTATAAAGATCCCAGGCGTTTCCCCCTGGTAGCTCCCTCGTGCGCTCTCCTGTTC
CTGCCTTTCGGTTTACCGGTGTCATTCCGCTGTTATGGCCGCGTTTGTCTCATTCCACGCCTGACACTCAGTTCC GGGTAGGCAGTTCGCTCCAAGCTGGACTGTATGCACGAACCCCCCGTTCAGTCCGACTACCACGCCCGTTCCGGT AACTATCAACTTGAGTCCAACCCGGAAAGACACGACAAATCGCCAGTGGCGGTAGCCATTGGTAACTGAGATGTG CGAGAGATTTATCTGGAGTTCTTGAAGTGGGGGCCTGAGTGCGGCTACACTGGAAGGACAGTTTAGGTGACTCGT CTCGCACAAGACAGTTACCACGGTTCATAAACTTGCCGGTTTAATGAACCTTCGAAAAACCACCTTGCCGGGTGG TTTTTTCTTTTCAAAGAAGATACGCGTTTACTCTTCGCCTCGTCAATGAAGGGTCGCATTAATTCGATCCCGCGA AATTAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTTTGTTTAACTTTA AGAAGGAGATATACATATGCAGATCTTCGTTAAAACCCTGACCGGTAAAACCATCACCCTGGAAGTTGAACCGTC TGACACCATCGAAAACGTTAAAGCGAAAATCCAGGACAAAGAAGGTATCCCGCCGGACCAGCAGGAACTGATCTT CGCGGGTAAACAGCTGGAAGACGGTCGTACCCTGTCTGACTACAACATCCAGAAAGAATCTACCCTGCACCTGGT TCTGCAG
Gene of interest i corporation into the plasmid of the invention
Gene for lysostaphin was processed by restriction enzymes Ndel/Notl in CutSmart restriction Buffer (New England Biolabs, USA) for 1 hour at 37°C. The restriction mixture was separated using DNA electrophoresis. The DNA fragment of appropriate size was excised and isolated by isolation kit NucleoSpin Gel and PCR Clean-up (Macherey-Nagel). The DNA concentration was determined spectrophotometrically. The vector was linearised by the same procedure. The incorporation of the gene of interest was achieved via DNA ligation. Ten ng of the linearised insert was incubated with 100 ng of linearised vector plasmid of the invention in the presence of T4 DNA ligase for 12 hours at 16°C. The reaction product was used for heat-shock transformation of E. coli DH10β chemo-competent cells. Cultivation on agar plates with kanamycin selection was used to select cells carrying the plasmid. The presence of the gene of interest in the plasmid of the invention was confirmed by DNA sequencing.
Protein expression experiments
The plasmid of the invention containing lysostaphin gene coding sequence was used to transform production strain E. colt BL21 (DE3). 50 ng of plasmid DNA was added to chemo -competent cells, which were transformed by heat shock (42°C/60 sec.) in the water bath. Positive clones were selected using seeding on LB agar plates with kanamycin. Fifteen colonies from the Petri dish were transferred to a liquid LB medium with kanamycin (100 pg/ml) and glucose (1 %). The inoculum was cultivated at 37°C for 12 hours under constant agitation at 220 rpm. 1 ml of cell culture was used to inoculate 100 ml of sterile autoinduction media (composition for IL: 10 g Tryptone; 5 g Yeast extract; 3.3 g ammonium sulphate; 50 mM phosphate buffer pH 6.7; 0.5 g glucose; 2 g lactose). The cultivation was kept for 48 hours at 22°C under constant agitation at 220 rpm. The protein production was verified by protein SDS- PAGE electrophoresis and subsequent Coomasie Brilliant blue staining of whole cell lysates. For three proteins, the production rate was analysed in more detail.
Comparison of expression of lysostaphin.
The production of lysostaphin protein was compared with the commonly used laboratory expression plasmid pET. The result shows a comparison of the measured pellet optical density (McFarland 0.5 = 1.5 x 108 CFU/ml) between the new plasmid of the invention and pET28. The difference is most pronounced in the first 24 hours, when of the invention-Lysostaphin has a density of only 5 McFarland and pET28-Lysostaphin has a density of 13 McFarland. After 48 hours, the density of pET28- Lysostaphin increased to 22 McFarland and of the invention-Lysostaphin to 25 McFarland. The resulting plasmid of the invention-Lysostaphin pellet had a lower yield (8.25 g/L) than pET28- Lysostaphin (13.75 g/L). However, the final protein production was 4.96x higher with the pUbExlOO- Lysostaphin. Due to the relatively higher production of protein per unit of biomass, the plasmid of the invention improves the subsequent purification process and makes its production significantly more efficient.
Claims
1. An expression vector which comprises, in the given order: an ORI sequence; a gene encoding ubiquitin as a leader protein; multiple cloning site; at least one affinity tag; TEV site; and at least one gene for selection antibiotic resistance; wherein the ORI sequence has a sequence SEQ ID NO: 2:
5 ' -GCAGACAGTTTTATTGTTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGT
AGAAAAGAGCGCATGCCGTGAGACTGCGGCGGGCGTTACCGGCTCACAAATAACGGGATACGCAGGCAGTGCTCA
AATCAGGAAGGACCGGAAAAAGGATGCGGCGTAGCCGTTTTTCCATAGGCTCCGCCCCCCTGACAAGCATCACGA
AATCTGACGCTCAAATCAGTGGCGGCGAAACCCGACAGGACTATAAAGATCCCAGGCGTTTCCCCCTGGTAGCTC
CCTCGTGCGCTCTCCTGTTCCTGCCTTTCGGTTTACCGGTGTCATTCCGCTGTTATGGCCGCGTTTGTCTCATTC
CACGCCTGACACTCAGTTCCGGGTAGGCAGTTCGCTCCAAGCTGGACTGTATGCACGAACCCCCCGTTCAGTCCG
ACTACCACGCCCGTTCCGGTAACTATCAACTTGAGTCCAACCCGGAAAGACACGACAAATCGCCAGTGGCGGTAG
CCATTGGTAACTGAGATGTGCGAGAGATTTATCTGGAGTTCTTGAAGTGGGGGCCTGAGTGCGGCTACACTGGAA GGACAGTTTAGGTGACTCGTCTCGCACAAGACAGTTACCACGGTTCATAAACTTGCCGGTTTAATGAACCTTCGA
AAAACCACCTTGCCGGGTGGTTTTTTCTTTTCAAAGAAGATACGCGTTTACTCTTCGCCTCGTCAATGAAGGGTC GCATTAAT-3 ' .
2. The expression vector according to claim 1, wherein the gene for selection antibiotic resistance is a kanamycin resistance gene having the sequence SEQ ID NO: 12:
5 ' -TTAGAAAAACTCATCCAGCATCAGGTGAAATTGCAGCTTATTCATATCCGGGTTGTCGATGCCATATTTTTG
AAACAGACGCTTTTGCAGGCTCGGGCTGAACTCGCCCAGGCAGTTCCACAGAATCGCCAGATCTTGGTAACGGTC
CGCGATGCCAACACGACCCACATCAATGCAGCCGATCAGTTTACCCTCGTCAAAGATCAGGTTATCCAGGCTGAA
GTCGCCGTGGGTAACCACGCTATCCGGGCTAAACGGCAGCAGTTTGTGCATTTCCTTCCAAACCTGCTCCACCGG
CCAGCCGTTACGTTCATCGTCGAAATCGCTCGCGTCAACCAGACCGTTGTTCATACGGCTTTGCGCCTGCGCCAG
ACGAAAAACACGGTCGCTGTTGAACGGGCAGTTGCACACCGGAATGCTGTGCAGACGACGCAGAAAAACCGCCAG
CGCATCCACGATGTTCTCGCCGCTGTCCGGGTATTCCTCCAGCACTTGGAACGCGGTTTTACCCGGAATCGCGGT
GGTCAGCAGCCACGCATCATCCGGGGTACGAATAAAGTGCTTGATGGTCGGCAGCGGCATGAACTCGGTCAGCCA
GTTCAGACGAACCATCTCATCGGTCACGTCGTTCGCCACGCTGCCTTTACCGTGCTTCAGGAACAGCTCCGGCGC
ATCCGGCTTGCCATACAGACGGTAGATGGTCGCGCCGCTCTGACCCACGTTGTCACGCGCCCACTTATAACCATA CAGATCCGCATCCATGTTGCTATTCAGACGCGGACGGCTACAGCTGGTCTCACGTTGAATGTGCAT- 3 '
3. The expression vector according to any one of claims 1 to 2, wherein the gene encoding ubiquitin has the sequence SEQ ID NO: 3:
5 ' -ATGCAGATCTTCGTTAAAACCCTGACCGGTAAAACCATCACCCTGGAAGTTGAACCGTCTGACACCATCGAA
AACGTTAAAGCGAAAATCCAGGACAAAGAAGGTATCCCGCCGGACCAGCAGGAACTGATCTTCGCGGGTAAACAG
CTGGAAGACGGTCGTACCCTGTCTGACTACAACATCCAGAAAGAATCTACCCTGCACCTGGTTCTGCAGCTCGAG TCTGCC-3 '
4. The expression vector according to claim 1, deposited with the German Collection of Microorganisms and Cell Cultures under the Accession No. DSM 34045 and having the DNA sequence SEQ ID NO: 1: CTCGAGTCTGCCCACCACCACCACTCTGGTCACCACCACACGGGTCACCACCACCACTCTGGTTCTCACCACCAC TCTGGTGCCGAAAACCTGTACTTCCAGTCTGGTTCTGGTACCATGGTTGGATCCGCTGCAGGAGCTCAAGCTTGC GGCCGCAGTCGACCACCACCACCACCACCACTGAGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGC TGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAA AGGAGGAACTATATCCGGATGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCAT GAATTAATTCTTAGAAAAACTCATCCAGCATCAGGTGAAATTGCAGCTTATTCATATCCGGGTTGTCGATGCCAT ATTTTTGAAACAGACGCTTTTGCAGGCTCGGGCTGAACTCGCCCAGGCAGTTCCACAGAATCGCCAGATCTTGGT AACGGTCCGCGATGCCAACACGACCCACATCAATGCAGCCGATCAGTTTACCCTCGTCAAAGATCAGGTTATCCA GGCTGAAGTCGCCGTGGGTAACCACGCTATCCGGGCTAAACGGCAGCAGTTTGTGCATTTCCTTCCAAACCTGCT CCACCGGCCAGCCGTTACGTTCATCGTCGAAATCGCTCGCGTCAACCAGACCGTTGTTCATACGGCTTTGCGCCT GCGCCAGACGAAAAACACGGTCGCTGTTGAACGGGCAGTTGCACACCGGAATGCTGTGCAGACGACGCAGAAAAA CCGCCAGCGCATCCACGATGTTCTCGCCGCTGTCCGGGTATTCCTCCAGCACTTGGAACGCGGTTTTACCCGGAA TCGCGGTGGTCAGCAGCCACGCATCATCCGGGGTACGAATAAAGTGCTTGATGGTCGGCAGCGGCATGAACTCGG TCAGCCAGTTCAGACGAACCATCTCATCGGTCACGTCGTTCGCCACGCTGCCTTTACCGTGCTTCAGGAACAGCT CCGGCGCATCCGGCTTGCCATACAGACGGTAGATGGTCGCGCCGCTCTGACCCACGTTGTCACGCGCCCACTTAT AACCATACAGATCCGCATCCATGTTGCTATTCAGACGCGGACGGCTACAGCTGGTCTCACGTTGAATGTGCATAA GAG C C CT T GT AT T ACT GT T T AT GTAAGCAGACAGT T T T AT T GT T CAT GAG CAAAAT C C CT T AAC GT GAGT T T T C G TTCCACTGAGCGTCAGACCCCGTAGAAAAGAGCGCATGCCGTGAGACTGCGGCGGGCGTTACCGGCTCACAAATA ACGGGATACGCAGGCAGTGCTCAAATCAGGAAGGACCGGAAAAAGGATGCGGCGTAGCCGTTTTTCCATAGGCTC CGCCCCCCTGACAAGCATCACGAAATCTGACGCTCAAATCAGTGGCGGCGAAACCCGACAGGACTATAAAGATCC CAGGCGTTTCCCCCTGGTAGCTCCCTCGTGCGCTCTCCTGTTCCTGCCTTTCGGTTTACCGGTGTCATTCCGCTG TTATGGCCGCGTTTGTCTCATTCCACGCCTGACACTCAGTTCCGGGTAGGCAGTTCGCTCCAAGCTGGACTGTAT GCACGAACCCCCCGTTCAGTCCGACTACCACGCCCGTTCCGGTAACTATCAACTTGAGTCCAACCCGGAAAGACA CGACAAATCGCCAGTGGCGGTAGCCATTGGTAACTGAGATGTGCGAGAGATTTATCTGGAGTTCTTGAAGTGGGG GCCTGAGTGCGGCTACACTGGAAGGACAGTTTAGGTGACTCGTCTCGCACAAGACAGTTACCACGGTTCATAAAC TTGCCGGTTTAATGAACCTTCGAAAAACCACCTTGCCGGGTGGTTTTTTCTTTTCAAAGAAGATACGCGTTTACT CTTCGCCTCGTCAATGAAGGGTCGCATTAATTCGATCCCGCGAAATTAATACGACTCACTATAGGGGAATTGTGA GCGGATAACAATTCCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACATATGCAGATCTTCGTTA AAACCCTGACCGGTAAAACCATCACCCTGGAAGTTGAACCGTCTGACACCATCGAAAACGTTAAAGCGAAAATCC AGGACAAAGAAGGTATCCCGCCGGACCAGCAGGAACTGATCTTCGCGGGTAAACAGCTGGAAGACGGTCGTACCC TGTCTGACTACAACATCCAGAAAGAATCTACCCTGCACCTGGTTCTGCAG .
5. The expression vector according to claim 1, further comprising a nucleotide sequence optimized for E. coli encoding Apxla and having the sequence SEQ ID NO: 18:
CTCGAGTCTGCCCACCACCACCACTCTGGTCACCACCACACGGGTCACCACCACCACTCTGGTTCTCACCACCAC TCTGGTGCCGAAAACCTGTACTTCCAGTCTGGTTCTGGTACCATGGGATCCTCTGTTGAAGAAATTATCGGTAGT AATCGTAAAGACAAATTCTTTGGTAGTCGCTTTACCGATATTTTCCATGGTGCGAAAGGCGATGATGAAATCTAC GGTAATGACGGCCACGATATCTTATACGGAGACGACGGTAATGATGTAATCCATGGCGGTGACGGTAACGACCAT
CTTGTTGGTGGTAACGGAAACGACCGATTAATCGGCGGAAAAGGTAATAATTTCCTTAATGGCGGTGATGGTGAC GATGAGTTGCAGGTCTTTGAGGGTCAATACAACGTATTATTAGGTGGTGCGGGTAATGACATTCTGTATGGCAGC GATGGTACTAACTTATTTGACGGTGGTGTAGGCAATGACAAAATCTACGGTGGTTTAGGTAAGGATATTTATCGC TACAGTAAGGAGTACGGTCGTCATATCATTATTGAGAAAGGCGGTGATGATGATACGTTATTGTTATCGGATCTT AGTTTTAAAGATGTAGGATTTATCAGAATCGGTGATGATCTTCTTGTGAATAAAAGAATCGGAGGAACACTGTAT TACCATGAAGATTACAATGGGAATGCGCTCACGATTAAAGATTGGTTCAAGGAAGGTAAGCTTTCTAGATAATGA GCGGCCGCAGTCGACCACCACCACCACCACCACTGAGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTG GCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTG AAAGGAGGAACTATATCCGGATGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTC ATGAATTAATTCTTAGAAAAACTCATCCAGCATCAGGTGAAATTGCAGCTTATTCATATCCGGGTTGTCGATGCC ATATTTTTGAAACAGACGCTTTTGCAGGCTCGGGCTGAACTCGCCCAGGCAGTTCCACAGAATCGCCAGATCTTG GTAACGGTCCGCGATGCCAACACGACCCACATCAATGCAGCCGATCAGTTTACCCTCGTCAAAGATCAGGTTATC CAGGCTGAAGTCGCCGTGGGTAACCACGCTATCCGGGCTAAACGGCAGCAGTTTGTGCATTTCCTTCCAAACCTG CTCCACCGGCCAGCCGTTACGTTCATCGTCGAAATCGCTCGCGTCAACCAGACCGTTGTTCATACGGCTTTGCGC CTGCGCCAGACGAAAAACACGGTCGCTGTTGAACGGGCAGTTGCACACCGGAATGCTGTGCAGACGACGCAGAAA AACCGCCAGCGCATCCACGATGTTCTCGCCGCTGTCCGGGTATTCCTCCAGCACTTGGAACGCGGTTTTACCCGG
AATCGCGGTGGTCAGCAGCCACGCATCATCCGGGGTACGAATAAAGTGCTTGATGGTCGGCAGCGGCATGAACTC GGTCAGCCAGTTCAGACGAACCATCTCATCGGTCACGTCGTTCGCCACGCTGCCTTTACCGTGCTTCAGGAACAG CTCCGGCGCATCCGGCTTGCCATACAGACGGTAGATGGTCGCGCCGCTCTGACCCACGTTGTCACGCGCCCACTT ATAACCATACAGATCCGCATCCATGTTGCTATTCAGACGCGGACGGCTACAGCTGGTCTCACGTTGAATGTGCAT AACAC C C CT T GT AT TACT GT T TAT GT AAGCAGACAGT T T TAT T GT T CAT GAC CAAAAT C C CT T AAC GT GAGT T T T CGTTCCACTGAGCGTCAGACCCCGTAGAAAAGAGCGCATGCCGTGAGACTGCGGCGGGCGTTACCGGCTCACAAA TAACGGGATACGCAGGCAGTGCTCAAATCAGGAAGGACCGGAAAAAGGATGCGGCGTAGCCGTTTTTCCATAGGC TCCGCCCCCCTGACAAGCATCACGAAATCTGACGCTCAAATCAGTGGCGGCGAAACCCGACAGGACTATAAAGAT CCCAGGCGTTTCCCCCTGGTAGCTCCCTCGTGCGCTCTCCTGTTCCTGCCTTTCGGTTTACCGGTGTCATTCCGC TGTTATGGCCGCGTTTGTCTCATTCCACGCCTGACACTCAGTTCCGGGTAGGCAGTTCGCTCCAAGCTGGACTGT ATGCACGAACCCCCCGTTCAGTCCGACTACCACGCCCGTTCCGGTAACTATCAACTTGAGTCCAACCCGGAAAGA CACGACAAATCGCCAGTGGCGGTAGCCATTGGTAACTGAGATGTGCGAGAGATTTATCTGGAGTTCTTGAAGTGG GGGCCTGAGTGCGGCTACACTGGAAGGACAGTTTAGGTGACTCGTCTCGCACAAGACAGTTACCACGGTTCATAA ACTTGCCGGTTTAATGAACCTTCGAAAAACCACCTTGCCGGGTGGTTTTTTCTTTTCAAAGAAGATACGCGTTTA CTCTTCGCCTCGTCAATGAAGGGTCGCATTAATTCGATCCCGCGAAATTAATACGACTCACTATAGGGGAATTGT GAGCGGATAACAATTCCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACATATGCAGATCTTCGT
TAAAACCCTGACCGGTAAAACCATCACCCTGGAAGTTGAACCGTCTGACACCATCGAAAACGTTAAAGCGAAAAT CCAGGACAAAGAAGGTATCCCGCCGGACCAGCAGGAACTGATCTTCGCGGGTAAACAGCTGGAAGACGGTCGTAC CCTGTCTGACTACAACATCCAGAAAGAATCTACCCTGCACCTGGTTCTGCAG
6. The expression vector according to claim 1, further comprising a nucleotide sequence optimized for E. coli encoding Bst 5.9 and having the sequence SEQ ID NO: 19:
CTCGAGTCTGCCCACCACCACCACTCTGGTCACCACCACACGGGTCACCACCACCACTCTGGTTCTCACCACCAC TCTGGTGCCGAAAACCTGTACTTCCAGTCTGGTTCTGGTACCATGGTTGGATCCCTGCTGCACGAGTTTGGTCTG
CTGGAAAGCCCGAAAGCGCTGGAAGAGGCGCCGTGGCCGCCGCCGGAGGGTGCGTTCGTGGGCTTCGTTCTGAGC
CGTAAAGAGCCGATGTGGGCGGATCTGCTGGCGCTGGCGGCGGCGCGTGGTGGCCGTGTGCACCGTGCGCCGGAA
CCGTACAAAGCGCTGCGTGACCTGAAAGAAGCGCGTGGTCTGCTGGCGAAGGACCTGAGCGTGCTGGCGCTGCGT
GAGGGCCTGGGTCTGCCGCCGGGCGATGACCCGATGCTGCTGGCGTACCTGCTGGACCCGAGCAACACCACCCCG
GAAGGTGTTGCGCGTCGTTACGGCGGTGAATGGACCGAAGAGGCGGGCGAACGTGCGGCGCTGAGCGAACGTCTG
TTCGCGAACCTGTGGGGTCGTCTGGAGGGTGAAGAGCGTCTGCTGTGGCTGTACCGTGAGGTTGAACGTCCGCTG
AGCGCGGTTCTGGCGCACATGGAGGCGACCGGTGTTCGTCTGGATGTTGCGTATCTGCGTGCGCTGAGCCTGGAA
GTGGCGGAAGAGATCGCGCGTCTGGAAGCGGAAGTGTTCCGTCTGGCGGGTCACCCGTTTAACCTGAACAGCCGT
GATCAACTGGAACGTGTTCTGTTCGATGAACTGGGCCTGCCGGCGATTGGCAAAACCGAAAAAACCGGCAAGCGT
AGCACCAGCGCGGCGGTGCTGGAAGCGCTGCGTGAAGCGCACCCGATTGTTGAAAAAATTCTGCAATACCGTGAG
CTGACCAAGCTGAAAAGCACCTACATTGAGGGTCTGCTGAAGGTTGTGCGTCCGGATACCAAGAAAGTTCACACC
CGTTTTAACCAGACCGCGACCGCGACCGGTCGTCTGAGCAGCAGCGACCCGAACCTGCAAAACATCCCGGTGCGT
ACCCCGCTGGGTCAGCGTATTCGTCGTGCGTTTATCGCGggtGAGGGTTGGCTGCTGGTTGCGCTGGATTACAGC
CAGATCGAACTGCGTGTTCTGGCGCACCTGAGCGGTGACGAGAACCTGATTCGTGTGTTCCAGGAAGGCCGTGAC
ATCCACACCGAGACCGCGAGCTGGATGTTTGGTGTGCCGCGTGAGGCGGTTGACCCGCTGATGCGTCGTGCGGCG
AAGACCATCAACTTTGGCGTTCTGTACGGTATGAGCGCGCACCGTCTGAGCCAAGAACTGGCGATCCCGTATGAG
GAAGCGCAAGCGTTTATCGAGCGTTATTTCCAGAGCTTCCCGAAAGTTCGTGCGTGGATCGAGAAGACCCTGGAA
GAGGGCCGTCGTCGTGGTTACGTGGAGACCCTGTTTGGTCGTCGTCGTTACGTGCCGGACCTGGAGGCGCGTGTT
AAGAGCGTGCGTGAGGCGGCGGAACGTATGGCGTTTAACATGCCGGTTCAAGGCACCGCGGCGGATCTGATGAAG agcGCGATGGTGAAGCTGTTCCCGCGTCTGGAAGAAATGGGTGCGCGTATGCTGCTGCAAGTTCACGATGAGCTG
GTTCTGGAAGCGCCGAAGGAACGTGCGGAAGCGGTTGCGCGTCTGGCGAAGGAAGTGATGGAAGGCGTGTATCCG
CTGGCGGTTCCGCTGGAAGTTGAAGTTGGTATTGGCGAGGATTGGCTGAGCGCGAAAGAGGTCGACTAAGCGGCC
GCAGTCGACCACCACCACCACCACCACTGAGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCT
GCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGA
GGAACTATATCCGGATGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAAT
TAATTCTTAGAAAAACTCATCCAGCATCAGGTGAAATTGCAGCTTATTCATATCCGGGTTGTCGATGCCATATTT
TTGAAACAGACGCTTTTGCAGGCTCGGGCTGAACTCGCCCAGGCAGTTCCACAGAATCGCCAGATCTTGGTAACG
GTCCGCGATGCCAACACGACCCACATCAATGCAGCCGATCAGTTTACCCTCGTCAAAGATCAGGTTATCCAGGCT
GAAGTCGCCGTGGGTAACCACGCTATCCGGGCTAAACGGCAGCAGTTTGTGCATTTCCTTCCAAACCTGCTCCAC
CGGCCAGCCGTTACGTTCATCGTCGAAATCGCTCGCGTCAACCAGACCGTTGTTCATACGGCTTTGCGCCTGCGC
CAGACGAAAAACACGGTCGCTGTTGAACGGGCAGTTGCACACCGGAATGCTGTGCAGACGACGCAGAAAAACCGC
CAGCGCATCCACGATGTTCTCGCCGCTGTCCGGGTATTCCTCCAGCACTTGGAACGCGGTTTTACCCGGAATCGC
GGTGGTCAGCAGCCACGCATCATCCGGGGTACGAATAAAGTGCTTGATGGTCGGCAGCGGCATGAACTCGGTCAG
CCAGTTCAGACGAACCATCTCATCGGTCACGTCGTTCGCCACGCTGCCTTTACCGTGCTTCAGGAACAGCTCCGG
CGCATCCGGCTTGCCATACAGACGGTAGATGGTCGCGCCGCTCTGACCCACGTTGTCACGCGCCCACTTATAACC
ATACAGATCCGCATCCATGTTGCTATTCAGACGCGGACGGCTACAGCTGGTCTCACGTTGAATGTGCATAACACC
CCTTGTATTACTGTTTATGTAAGCAGACAGTTTTATTGTTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCC
ACTGAGCGTCAGACCCCGTAGAAAAGAGCGCATGCCGTGAGACTGCGGCGGGCGTTACCGGCTCACAAATAACGG
GATACGCAGGCAGTGCTCAAATCAGGAAGGACCGGAAAAAGGATGCGGCGTAGCCGTTTTTCCATAGGCTCCGCC
CCCCTGACAAGCATCACGAAATCTGACGCTCAAATCAGTGGCGGCGAAACCCGACAGGACTATAAAGATCCCAGG
CGTTTCCCCCTGGTAGCTCCCTCGTGCGCTCTCCTGTTCCTGCCTTTCGGTTTACCGGTGTCATTCCGCTGTTAT
GGCCGCGTTTGTCTCATTCCACGCCTGACACTCAGTTCCGGGTAGGCAGTTCGCTCCAAGCTGGACTGTATGCAC
GAACCCCCCGTTCAGTCCGACTACCACGCCCGTTCCGGTAACTATCAACTTGAGTCCAACCCGGAAAGACACGAC
AAATCGCCAGTGGCGGTAGCCATTGGTAACTGAGATGTGCGAGAGATTTATCTGGAGTTCTTGAAGTGGGGGCCT
GAGTGCGGCTACACTGGAAGGACAGTTTAGGTGACTCGTCTCGCACAAGACAGTTACCACGGTTCATAAACTTGC
CGGTTTAATGAACCTTCGAAAAACCACCTTGCCGGGTGGTTTTTTCTTTTCAAAGAAGATACGCGTTTACTCTTC
GCCTCGTCAATGAAGGGTCGCATTAATTCGATCCCGCGAAATTAATACGACTCACTATAGGGGAATTGTGAGCGG
ATAACAATTCCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACATATGCAGATCTTCGTTAAAAC
CCTGACCGGTAAAACCATCACCCTGGAAGTTGAACCGTCTGACACCATCGAAAACGTTAAAGCGAAAATCCAGGA
CAAAGAAGGTATCCCGCCGGACCAGCAGGAACTGATCTTCGCGGGTAAACAGCTGGAAGACGGTCGTACCCTGTC TGACTACAACATCCAGAAAGAATCTACCCTGCACCTGGTTCTGCAG
7. The expression vector according to claim 1, further comprising a nucleotide sequence optimized for
E. coli encoding Lysostaphin and having the sequence SEQ ID NO: 20:
CTCGAGTCTGCCCACCACCACCACTCTGGTCACCACCACACGGGTCACCACCACCACTCTGGTTCTCACCACCAC
TCTGGTGCCGAAAACCTGTACTTCCAGTCTGGTTCTGGTACCATGGCGCACGAGCACAGCGCGCAGTGGCTGAAC
AACTACAAGAAGGGTTATGGTTATGGTCCGTATCCGCTGGGTATCAATGGTGGTATGCACTACGGCGTGGACTTC
TTTATGAACATCGGTACCCCGGTGAAGGCGATCAGCAGCGGCAAAATTGTTGAGGCGGGTTGGAGCAACTATGGT
GGCGGTAACCAGATCGGCCTGATTGAAAACGACGGTGTGCACCGTCAATGGTACATGCACCTGAGCAAGTATAAC
GTGAAAGTTGGCGATTACGTTAAGGCGGGTCAGATCATTGGCTGGAGCGGTAGCACCGGTTATAGCACCGCGCCG
CACCTGCACTTCCAGCGTATGGTGAACAGCTTTAGCAACAGCACCGCGCAAGACCCGATGCCGTTCCTGAAGAGC
GCGGGTTATGGTAAAGCGGGCGGATCCGTTACCCCGACCCCGAACACCGGCTGGAAGACCAACAAATACGGCACC
CTGTATAAAAGCGAGAGCGCGAGCTTCACCCCGAACACCGATATCATTACCCGTACCACCGGCCCGTTTCGTAGC
ATGCCGCAGAGCGGCGTGCTGAAGGCGGGTCAAACCATCCACTATGACGAAGTTATGAAACAGGATGGTCACGTG
TGGGTTGGTTACACCGGCAACAGCGGTCAACGTATTTATCTGCCGGTTCGCACCTGGAATAAAAGCACCAACACC
CTGGGCGTTCTGTGGGGTACCATCAAATAAGCGGCCGCAGTCGACCACCACCACCACCACCACTGAGATCCGGCT
GCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCC
TCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGATGCGGAACCCCTATTTGTTTATTT
TTCTAAATACATTCAAATATGTATCCGCTCATGAATTAATTCTTAGAAAAACTCATCCAGCATCAGGTGAAATTG
CAGCTTATTCATATCCGGGTTGTCGATGCCATATTTTTGAAACAGACGCTTTTGCAGGCTCGGGCTGAACTCGCC
CAGGCAGTTCCACAGAATCGCCAGATCTTGGTAACGGTCCGCGATGCCAACACGACCCACATCAATGCAGCCGAT
CAGTTTACCCTCGTCAAAGATCAGGTTATCCAGGCTGAAGTCGCCGTGGGTAACCACGCTATCCGGGCTAAACGG
CAGCAGTTTGTGCATTTCCTTCCAAACCTGCTCCACCGGCCAGCCGTTACGTTCATCGTCGAAATCGCTCGCGTC
AACCAGACCGTTGTTCATACGGCTTTGCGCCTGCGCCAGACGAAAAACACGGTCGCTGTTGAACGGGCAGTTGCA
CACCGGAATGCTGTGCAGACGACGCAGAAAAACCGCCAGCGCATCCACGATGTTCTCGCCGCTGTCCGGGTATTC
CTCCAGCACTTGGAACGCGGTTTTACCCGGAATCGCGGTGGTCAGCAGCCACGCATCATCCGGGGTACGAATAAA
GTGCTTGATGGTCGGCAGCGGCATGAACTCGGTCAGCCAGTTCAGACGAACCATCTCATCGGTCACGTCGTTCGC
CACGCTGCCTTTACCGTGCTTCAGGAACAGCTCCGGCGCATCCGGCTTGCCATACAGACGGTAGATGGTCGCGCC
GCTCTGACCCACGTTGTCACGCGCCCACTTATAACCATACAGATCCGCATCCATGTTGCTATTCAGACGCGGACG
GCTACAGCTGGTCTCACGTTGAATGTGCATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAGTTTTATTGT
TCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGAGCGCATGCCGTG
AGACTGCGGCGGGCGTTACCGGCTCACAAATAACGGGATACGCAGGCAGTGCTCAAATCAGGAAGGACCGGAAAA
AGGATGCGGCGTAGCCGTTTTTCCATAGGCTCCGCCCCCCTGACAAGCATCACGAAATCTGACGCTCAAATCAGT
GGCGGCGAAACCCGACAGGACTATAAAGATCCCAGGCGTTTCCCCCTGGTAGCTCCCTCGTGCGCTCTCCTGTTC
CTGCCTTTCGGTTTACCGGTGTCATTCCGCTGTTATGGCCGCGTTTGTCTCATTCCACGCCTGACACTCAGTTCC
GGGTAGGCAGTTCGCTCCAAGCTGGACTGTATGCACGAACCCCCCGTTCAGTCCGACTACCACGCCCGTTCCGGT
AACTATCAACTTGAGTCCAACCCGGAAAGACACGACAAATCGCCAGTGGCGGTAGCCATTGGTAACTGAGATGTG
CGAGAGATTTATCTGGAGTTCTTGAAGTGGGGGCCTGAGTGCGGCTACACTGGAAGGACAGTTTAGGTGACTCGT
CTCGCACAAGACAGTTACCACGGTTCATAAACTTGCCGGTTTAATGAACCTTCGAAAAACCACCTTGCCGGGTGG
TTTTTTCTTTTCAAAGAAGATACGCGTTTACTCTTCGCCTCGTCAATGAAGGGTCGCATTAATTCGATCCCGCGA
AATTAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTTTGTTTAACTTTA
AGAAGGAGATATACATATGCAGATCTTCGTTAAAACCCTGACCGGTAAAACCATCACCCTGGAAGTTGAACCGTC
TGACACCATCGAAAACGTTAAAGCGAAAATCCAGGACAAAGAAGGTATCCCGCCGGACCAGCAGGAACTGATCTT
CGCGGGTAAACAGCTGGAAGACGGTCGTACCCTGTCTGACTACAACATCCAGAAAGAATCTACCCTGCACCTGGT TCTGCAG
8. DNA sequence
5 ' -GCAGACAGTTTTATTGTTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGT
AGAAAAGAGCGCATGCCGTGAGACTGCGGCGGGCGTTACCGGCTCACAAATAACGGGATACGCAGGCAGTGCTCA
AATCAGGAAGGACCGGAAAAAGGATGCGGCGTAGCCGTTTTTCCATAGGCTCCGCCCCCCTGACAAGCATCACGA
AATCTGACGCTCAAATCAGTGGCGGCGAAACCCGACAGGACTATAAAGATCCCAGGCGTTTCCCCCTGGTAGCTC
CCTCGTGCGCTCTCCTGTTCCTGCCTTTCGGTTTACCGGTGTCATTCCGCTGTTATGGCCGCGTTTGTCTCATTC
CACGCCTGACACTCAGTTCCGGGTAGGCAGTTCGCTCCAAGCTGGACTGTATGCACGAACCCCCCGTTCAGTCCG
ACTACCACGCCCGTTCCGGTAACTATCAACTTGAGTCCAACCCGGAAAGACACGACAAATCGCCAGTGGCGGTAG
CCATTGGTAACTGAGATGTGCGAGAGATTTATCTGGAGTTCTTGAAGTGGGGGCCTGAGTGCGGCTACACTGGAA
GGACAGTTTAGGTGACTCGTCTCGCACAAGACAGTTACCACGGTTCATAAACTTGCCGGTTTAATGAACCTTCGA
AAAACCACCTTGCCGGGTGGTTTTTTCTTTTCAAAGAAGATACGCGTTTACTCTTCGCCTCGTCAATGAAGGGTC GCATTAAT-3 ' (SEQ ID NO: 2).
9. Use of DNA sequence as ORI sequence in expression plasmids, wherein the DNA sequence is:
5 ' -GCAGACAGTTTTATTGTTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGT
AGAAAAGAGCGCATGCCGTGAGACTGCGGCGGGCGTTACCGGCTCACAAATAACGGGATACGCAGGCAGTGCTCA
AATCAGGAAGGACCGGAAAAAGGATGCGGCGTAGCCGTTTTTCCATAGGCTCCGCCCCCCTGACAAGCATCACGA
AATCTGACGCTCAAATCAGTGGCGGCGAAACCCGACAGGACTATAAAGATCCCAGGCGTTTCCCCCTGGTAGCTC
CCTCGTGCGCTCTCCTGTTCCTGCCTTTCGGTTTACCGGTGTCATTCCGCTGTTATGGCCGCGTTTGTCTCATTC
CACGCCTGACACTCAGTTCCGGGTAGGCAGTTCGCTCCAAGCTGGACTGTATGCACGAACCCCCCGTTCAGTCCG
ACTACCACGCCCGTTCCGGTAACTATCAACTTGAGTCCAACCCGGAAAGACACGACAAATCGCCAGTGGCGGTAG
CCATTGGTAACTGAGATGTGCGAGAGATTTATCTGGAGTTCTTGAAGTGGGGGCCTGAGTGCGGCTACACTGGAA
GGACAGTTTAGGTGACTCGTCTCGCACAAGACAGTTACCACGGTTCATAAACTTGCCGGTTTAATGAACCTTCGA
AAAACCACCTTGCCGGGTGGTTTTTTCTTTTCAAAGAAGATACGCGTTTACTCTTCGCCTCGTCAATGAAGGGTC
GCATTAAT-3 ' (SEQ ID NO: 2).
10. A method of producing a recombinant protein in a heterologous prokaryotic host cell, said method comprising the steps of: inserting the coding sequence of the protein to be produced into a plasmid according to the invention, transforming the plasmid into a prokaryotic host cell, culturing and harvesting the prokaryotic host cells, disintegrating the prokaryotic host cells to release the produced recombinant protein, optionally purifying the produced recombinant protein, and optionally cleaving the leader protein from the produced recombinant protein.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| PCT/CZ2022/050100 WO2023193837A1 (en) | 2022-10-03 | 2022-10-03 | Expression vector for production of recombinant proteins in prokaryotic host cells |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| PCT/CZ2022/050100 WO2023193837A1 (en) | 2022-10-03 | 2022-10-03 | Expression vector for production of recombinant proteins in prokaryotic host cells |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO2023193837A1 true WO2023193837A1 (en) | 2023-10-12 |
Family
ID=84053320
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/CZ2022/050100 WO2023193837A1 (en) | 2022-10-03 | 2022-10-03 | Expression vector for production of recombinant proteins in prokaryotic host cells |
Country Status (1)
| Country | Link |
|---|---|
| WO (1) | WO2023193837A1 (en) |
-
2022
- 2022-10-03 WO PCT/CZ2022/050100 patent/WO2023193837A1/en unknown
Non-Patent Citations (6)
| Title |
|---|
| DATABASE EMBL [online] 23 April 2007 (2007-04-23), "Corynebacterium renale plasmid pCR2, complete sequence.", XP002809363, retrieved from EBI accession no. EM_STD:EF488047 Database accession no. EF488047 * |
| EGUIA FARA AMELIA PRIMELLES ET AL: "A new vector for heterologous gene expression in Escherichia coli with increased stability in the absence of antibiotic", PLASMID, NEW YORK,NY, US, vol. 98, 5 September 2018 (2018-09-05), pages 22 - 30, XP085496317, ISSN: 0147-619X, DOI: 10.1016/J.PLASMID.2018.08.004 * |
| GERMÁN L. ROSANO ET AL: "Recombinant protein expression in Escherichia coli: advances and challenges", FRONTIERS IN MICROBIOLOGY, vol. 5, 17 April 2014 (2014-04-17), XP055474138, DOI: 10.3389/fmicb.2014.00172 * |
| LOZANO TEROL GEMA ET AL: "Impact of the Expression System on Recombinant Protein Production in Escherichia coli BL21", FRONTIERS IN MICROBIOLOGY, vol. 12, 21 June 2021 (2021-06-21), XP093048970, DOI: 10.3389/fmicb.2021.682001 * |
| ROGOV VVROZENKNOP AROGOVA NYLOHR FTIKOLE SJARAVINE VGIINTERT PDIKIC IDOTSCH V: "A universal expression tag for structural and functional studies of proteins", CHEMBIOCHEM, vol. 13, no. 7, 7 May 2012 (2012-05-07), pages 959 - 63 |
| WALIA RUPALI ET AL: "Development of expression vectors for Escherichia coli based on the pCR2 replicon", MICROBIAL CELL FACTORIES, SPRINGER, vol. 6, no. 1, 10 May 2007 (2007-05-10), pages 14, XP021024081, ISSN: 1475-2859, DOI: 10.1186/1475-2859-6-14 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| EP0946711B1 (en) | Improved expression vectors | |
| CA2619989C (en) | Regulation of heterologous recombinant protein expression in methylotrophic and methanotrophic bacteria | |
| DK2689015T3 (en) | A process for the production of secreted polypeptides | |
| CN108220288B (en) | Polynucleotide, transformant and application thereof | |
| US8394937B2 (en) | Expression system | |
| TWI450965B (en) | Vector comprising mannose promoter and mannose promoter | |
| CN111117942B (en) | Genetic engineering bacterium for producing lincomycin and construction method and application thereof | |
| CN101978057A (en) | Method of modifying target region in host DNA and selectable marker cassette | |
| HU201116B (en) | Process for producing plasmids having uncontrolled replication conduct under given circumstances | |
| JP6071878B2 (en) | Inducible promoter regulation | |
| CA2467142C (en) | Improved method for the recombinant production of polypeptides | |
| CN109321618B (en) | Method for improving erythromycin yield through saccharopolyspora erythraea SACE _5717 gene | |
| US20120196323A1 (en) | Fermentation Process | |
| WO2023193837A1 (en) | Expression vector for production of recombinant proteins in prokaryotic host cells | |
| US11203760B2 (en) | Gene therapy DNA vector GDTT1.8NAS12 and the method for obtaining thereof | |
| US6218145B1 (en) | Bacterial expression systems based on plastic or mitochondrial promoter combinations | |
| CN117187210B (en) | Mutant Bst DNA polymerase large fragment and preparation method thereof | |
| KR102166288B1 (en) | D-glutamate auxotrophic Escherichia coli and method of producing target product | |
| KR101175725B1 (en) | Novel Gram-positive Bacteria Expression System | |
| JPH06105693A (en) | Dna fragment having base sequency coding esterase | |
| CN116003570A (en) | Recombinant hLIF mutant with improved thermal stability and preparation method and application thereof | |
| KR100411238B1 (en) | Novel Bacillus sp. for the Stable Expression of Foreign Proteins | |
| Gholizadeh et al. | Induced expression of Eco RI endonuclease as an active maltose-binding fusion protein in Escherichia coli | |
| SOCOL et al. | Molecular cloning of ovine cDNA leptin gene | |
| JPS59162882A (en) | Plasmid |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| 121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 22799844 Country of ref document: EP Kind code of ref document: A1 |