CN113528574A - Signal peptide related sequence and application thereof in protein synthesis - Google Patents
Signal peptide related sequence and application thereof in protein synthesis Download PDFInfo
- Publication number
- CN113528574A CN113528574A CN202110864773.4A CN202110864773A CN113528574A CN 113528574 A CN113528574 A CN 113528574A CN 202110864773 A CN202110864773 A CN 202110864773A CN 113528574 A CN113528574 A CN 113528574A
- Authority
- CN
- China
- Prior art keywords
- vitro
- sequence
- protein
- signal peptide
- cell
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 230000014616 translation Effects 0.000 title claims abstract description 103
- 108010076504 Protein Sorting Signals Proteins 0.000 title claims abstract description 85
- 238000001243 protein synthesis Methods 0.000 title claims abstract description 81
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 127
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 115
- 102000039446 nucleic acids Human genes 0.000 claims abstract description 41
- 108020004707 nucleic acids Proteins 0.000 claims abstract description 41
- 150000007523 nucleic acids Chemical class 0.000 claims abstract description 41
- 239000013598 vector Substances 0.000 claims abstract description 30
- 238000000338 in vitro Methods 0.000 claims description 103
- 210000004027 cell Anatomy 0.000 claims description 64
- 239000002773 nucleotide Substances 0.000 claims description 52
- 125000003729 nucleotide group Chemical group 0.000 claims description 52
- 230000015572 biosynthetic process Effects 0.000 claims description 28
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 25
- 238000000034 method Methods 0.000 claims description 23
- 210000005253 yeast cell Anatomy 0.000 claims description 19
- 241000235649 Kluyveromyces Species 0.000 claims description 13
- 238000006243 chemical reaction Methods 0.000 claims description 12
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 12
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 12
- 229920001184 polypeptide Polymers 0.000 claims description 10
- 241000238631 Hexapoda Species 0.000 claims description 9
- 230000001851 biosynthetic effect Effects 0.000 claims description 8
- 210000004978 chinese hamster ovary cell Anatomy 0.000 claims description 7
- 241000283973 Oryctolagus cuniculus Species 0.000 claims description 6
- 210000001995 reticulocyte Anatomy 0.000 claims description 6
- 230000035484 reaction time Effects 0.000 claims description 5
- 210000001236 prokaryotic cell Anatomy 0.000 claims description 4
- 238000011144 upstream manufacturing Methods 0.000 claims description 4
- 239000003153 chemical reaction reagent Substances 0.000 claims description 3
- 230000002194 synthesizing effect Effects 0.000 claims description 3
- 125000003275 alpha amino acid group Chemical group 0.000 claims 2
- 108091026890 Coding region Proteins 0.000 abstract description 39
- 230000014509 gene expression Effects 0.000 abstract description 21
- 230000000694 effects Effects 0.000 abstract description 9
- 238000010353 genetic engineering Methods 0.000 abstract description 4
- 238000000746 purification Methods 0.000 abstract description 4
- 235000018102 proteins Nutrition 0.000 description 83
- 102000040430 polynucleotide Human genes 0.000 description 64
- 108091033319 polynucleotide Proteins 0.000 description 64
- 239000002157 polynucleotide Substances 0.000 description 64
- 108020004414 DNA Proteins 0.000 description 42
- 102000053602 DNA Human genes 0.000 description 42
- 150000001413 amino acids Chemical class 0.000 description 26
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 description 23
- 239000013612 plasmid Substances 0.000 description 18
- 241001138401 Kluyveromyces lactis Species 0.000 description 15
- 108060001084 Luciferase Proteins 0.000 description 13
- 239000005089 Luciferase Substances 0.000 description 13
- 238000013519 translation Methods 0.000 description 13
- 235000001014 amino acid Nutrition 0.000 description 12
- 229940024606 amino acid Drugs 0.000 description 12
- 230000000295 complement effect Effects 0.000 description 10
- 239000000284 extract Substances 0.000 description 10
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 10
- 210000003527 eukaryotic cell Anatomy 0.000 description 9
- 102000016938 Catalase Human genes 0.000 description 8
- 108010053835 Catalase Proteins 0.000 description 8
- DRBBFCLWYRJSJZ-UHFFFAOYSA-N N-phosphocreatine Chemical compound OC(=O)CN(C)C(=N)NP(O)(O)=O DRBBFCLWYRJSJZ-UHFFFAOYSA-N 0.000 description 8
- 239000012634 fragment Substances 0.000 description 8
- 102000006602 glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 description 8
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 8
- 108091005957 yellow fluorescent proteins Proteins 0.000 description 8
- 102000052866 Amino Acyl-tRNA Synthetases Human genes 0.000 description 7
- 108700028939 Amino Acyl-tRNA Synthetases Proteins 0.000 description 7
- IGXWBGJHJZYPQS-SSDOTTSWSA-N D-Luciferin Chemical compound OC(=O)[C@H]1CSC(C=2SC3=CC=C(O)C=C3N=2)=N1 IGXWBGJHJZYPQS-SSDOTTSWSA-N 0.000 description 7
- CYCGRDQQIOGCKX-UHFFFAOYSA-N Dehydro-luciferin Natural products OC(=O)C1=CSC(C=2SC3=CC(O)=CC=C3N=2)=N1 CYCGRDQQIOGCKX-UHFFFAOYSA-N 0.000 description 7
- 108090000331 Firefly luciferases Proteins 0.000 description 7
- BJGNCJDXODQBOB-UHFFFAOYSA-N Fivefly Luciferin Natural products OC(=O)C1CSC(C=2SC3=CC(O)=CC=C3N=2)=N1 BJGNCJDXODQBOB-UHFFFAOYSA-N 0.000 description 7
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 7
- 102000004144 Green Fluorescent Proteins Human genes 0.000 description 7
- DDWFXDSYGUXRAY-UHFFFAOYSA-N Luciferin Natural products CCc1c(C)c(CC2NC(=O)C(=C2C=C)C)[nH]c1Cc3[nH]c4C(=C5/NC(CC(=O)O)C(C)C5CC(=O)O)CC(=O)c4c3C DDWFXDSYGUXRAY-UHFFFAOYSA-N 0.000 description 7
- 238000005119 centrifugation Methods 0.000 description 7
- 238000010276 construction Methods 0.000 description 7
- 239000005090 green fluorescent protein Substances 0.000 description 7
- 239000003550 marker Substances 0.000 description 7
- 229920001223 polyethylene glycol Polymers 0.000 description 7
- 239000013641 positive control Substances 0.000 description 7
- 239000000047 product Substances 0.000 description 7
- 238000011282 treatment Methods 0.000 description 7
- 238000005406 washing Methods 0.000 description 7
- 102000007469 Actins Human genes 0.000 description 6
- 108010085238 Actins Proteins 0.000 description 6
- 241000894006 Bacteria Species 0.000 description 6
- 102000004163 DNA-directed RNA polymerases Human genes 0.000 description 6
- 108090000626 DNA-directed RNA polymerases Proteins 0.000 description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 6
- 239000002202 Polyethylene glycol Substances 0.000 description 6
- 150000001875 compounds Chemical class 0.000 description 6
- 239000000203 mixture Substances 0.000 description 6
- SCVFZCLFOSHCOH-UHFFFAOYSA-M potassium acetate Chemical compound [K+].CC([O-])=O SCVFZCLFOSHCOH-UHFFFAOYSA-M 0.000 description 6
- LOGFVTREOLYCPF-KXNHARMFSA-N (2s,3r)-2-[[(2r)-1-[(2s)-2,6-diaminohexanoyl]pyrrolidine-2-carbonyl]amino]-3-hydroxybutanoic acid Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@H]1CCCN1C(=O)[C@@H](N)CCCCN LOGFVTREOLYCPF-KXNHARMFSA-N 0.000 description 5
- UBAVPHLFRVPRCI-CRBCFSCISA-N 2-[[(e)-2-carboxyethenyl]amino]-n-methoxy-2-oxoethanimine oxide Chemical compound CO\[N+]([O-])=C\C(=O)N\C=C\C(O)=O UBAVPHLFRVPRCI-CRBCFSCISA-N 0.000 description 5
- 108020004705 Codon Proteins 0.000 description 5
- 241000196324 Embryophyta Species 0.000 description 5
- 101710121765 Endo-1,4-beta-xylanase Proteins 0.000 description 5
- 108010073141 Hepatitis C virus glycoprotein E2 Proteins 0.000 description 5
- 102000008394 Immunoglobulin Fragments Human genes 0.000 description 5
- 108010021625 Immunoglobulin Fragments Proteins 0.000 description 5
- 102000004877 Insulin Human genes 0.000 description 5
- 108090001061 Insulin Proteins 0.000 description 5
- 108010078049 Interferon alpha-2 Proteins 0.000 description 5
- 102100040018 Interferon alpha-2 Human genes 0.000 description 5
- 102000003777 Interleukin-1 beta Human genes 0.000 description 5
- 108090000193 Interleukin-1 beta Proteins 0.000 description 5
- 102000016943 Muramidase Human genes 0.000 description 5
- 108010014251 Muramidase Proteins 0.000 description 5
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 description 5
- 108010071690 Prealbumin Proteins 0.000 description 5
- 102000007562 Serum Albumin Human genes 0.000 description 5
- 108010071390 Serum Albumin Proteins 0.000 description 5
- 102000009190 Transthyretin Human genes 0.000 description 5
- 241000209140 Triticum Species 0.000 description 5
- 235000021307 Triticum Nutrition 0.000 description 5
- 102000003425 Tyrosinase Human genes 0.000 description 5
- 108060008724 Tyrosinase Proteins 0.000 description 5
- 102000004139 alpha-Amylases Human genes 0.000 description 5
- 108090000637 alpha-Amylases Proteins 0.000 description 5
- 229940024171 alpha-amylase Drugs 0.000 description 5
- 230000003321 amplification Effects 0.000 description 5
- 230000008901 benefit Effects 0.000 description 5
- 238000002474 experimental method Methods 0.000 description 5
- 230000006870 function Effects 0.000 description 5
- 229940125396 insulin Drugs 0.000 description 5
- 239000004325 lysozyme Substances 0.000 description 5
- 229960000274 lysozyme Drugs 0.000 description 5
- 235000010335 lysozyme Nutrition 0.000 description 5
- 239000013642 negative control Substances 0.000 description 5
- 238000003199 nucleic acid amplification method Methods 0.000 description 5
- 239000002777 nucleoside Substances 0.000 description 5
- 239000002243 precursor Substances 0.000 description 5
- 238000012216 screening Methods 0.000 description 5
- 239000001226 triphosphate Substances 0.000 description 5
- 235000011178 triphosphate Nutrition 0.000 description 5
- -1 D-amino acids) Chemical class 0.000 description 4
- 241000588724 Escherichia coli Species 0.000 description 4
- 241000206602 Eukaryota Species 0.000 description 4
- 108091028043 Nucleic acid sequence Proteins 0.000 description 4
- 229930006000 Sucrose Natural products 0.000 description 4
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 4
- 210000004102 animal cell Anatomy 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 239000005720 sucrose Substances 0.000 description 4
- 230000009466 transformation Effects 0.000 description 4
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 3
- ZKHQWZAMYRWXGA-UHFFFAOYSA-N Adenosine triphosphate Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)C(O)C1O ZKHQWZAMYRWXGA-UHFFFAOYSA-N 0.000 description 3
- YWAQATDNEKZFFK-BYPYZUCNSA-N Gly-Gly-Ser Chemical compound NCC(=O)NCC(=O)N[C@@H](CO)C(O)=O YWAQATDNEKZFFK-BYPYZUCNSA-N 0.000 description 3
- 108010020764 Transposases Proteins 0.000 description 3
- 229940041514 candida albicans extract Drugs 0.000 description 3
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 description 3
- 229940079593 drug Drugs 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 230000001965 increasing effect Effects 0.000 description 3
- UEGPKNKPLBYCNK-UHFFFAOYSA-L magnesium acetate Chemical compound [Mg+2].CC([O-])=O.CC([O-])=O UEGPKNKPLBYCNK-UHFFFAOYSA-L 0.000 description 3
- 239000011654 magnesium acetate Substances 0.000 description 3
- 229940069446 magnesium acetate Drugs 0.000 description 3
- 235000011285 magnesium acetate Nutrition 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 229950007002 phosphocreatine Drugs 0.000 description 3
- 235000011056 potassium acetate Nutrition 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- 239000012138 yeast extract Substances 0.000 description 3
- 241000589158 Agrobacterium Species 0.000 description 2
- ANGAOPNEPIDLPO-XVYDVKMFSA-N Ala-His-Cys Chemical compound C[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)N[C@@H](CS)C(=O)O)N ANGAOPNEPIDLPO-XVYDVKMFSA-N 0.000 description 2
- OYJCVIGKMXUVKB-GARJFASQSA-N Ala-Leu-Pro Chemical compound C[C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N1CCC[C@@H]1C(=O)O)N OYJCVIGKMXUVKB-GARJFASQSA-N 0.000 description 2
- SPIPSJXLZVTXJL-ZLUOBGJFSA-N Asn-Cys-Ser Chemical compound NC(=O)C[C@H](N)C(=O)N[C@@H](CS)C(=O)N[C@@H](CO)C(O)=O SPIPSJXLZVTXJL-ZLUOBGJFSA-N 0.000 description 2
- CBWCQCANJSGUOH-ZKWXMUAHSA-N Asn-Val-Ala Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](C)C(O)=O CBWCQCANJSGUOH-ZKWXMUAHSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 241000233866 Fungi Species 0.000 description 2
- 108700028146 Genetic Enhancer Elements Proteins 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- GVNNAHIRSDRIII-AJNGGQMLSA-N Ile-Lys-Lys Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)O)N GVNNAHIRSDRIII-AJNGGQMLSA-N 0.000 description 2
- JZBVBOKASHNXAD-NAKRPEOUSA-N Ile-Val-Ser Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CO)C(=O)O)N JZBVBOKASHNXAD-NAKRPEOUSA-N 0.000 description 2
- 235000014663 Kluyveromyces fragilis Nutrition 0.000 description 2
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 2
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 2
- GZRABTMNWJXFMH-UVOCVTCTSA-N Leu-Thr-Thr Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O GZRABTMNWJXFMH-UVOCVTCTSA-N 0.000 description 2
- 102000043856 Leucine-tRNA ligase Human genes 0.000 description 2
- 108010071170 Leucine-tRNA ligase Proteins 0.000 description 2
- 102000003960 Ligases Human genes 0.000 description 2
- 108090000364 Ligases Proteins 0.000 description 2
- IEVXCWPVBYCJRZ-IXOXFDKPSA-N Lys-Thr-His Chemical compound NCCCC[C@H](N)C(=O)N[C@@H]([C@H](O)C)C(=O)N[C@H](C(O)=O)CC1=CN=CN1 IEVXCWPVBYCJRZ-IXOXFDKPSA-N 0.000 description 2
- 241000124008 Mammalia Species 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 241000288906 Primates Species 0.000 description 2
- 102000001253 Protein Kinase Human genes 0.000 description 2
- 108020004511 Recombinant DNA Proteins 0.000 description 2
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 2
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 2
- 241000283984 Rodentia Species 0.000 description 2
- 235000018368 Saccharomyces fragilis Nutrition 0.000 description 2
- SIEBDTCABMZCLF-XGEHTFHBSA-N Ser-Val-Thr Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O SIEBDTCABMZCLF-XGEHTFHBSA-N 0.000 description 2
- 108091081024 Start codon Proteins 0.000 description 2
- 101710137500 T7 RNA polymerase Proteins 0.000 description 2
- IMDMLDSVUSMAEJ-HJGDQZAQSA-N Thr-Leu-Asn Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(N)=O)C(O)=O IMDMLDSVUSMAEJ-HJGDQZAQSA-N 0.000 description 2
- BBPCSGKKPJUYRB-UVOCVTCTSA-N Thr-Thr-Leu Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(O)=O BBPCSGKKPJUYRB-UVOCVTCTSA-N 0.000 description 2
- 108020004566 Transfer RNA Proteins 0.000 description 2
- 102000008579 Transposases Human genes 0.000 description 2
- ISAKRJDGNUQOIC-UHFFFAOYSA-N Uracil Chemical compound O=C1C=CNC(=O)N1 ISAKRJDGNUQOIC-UHFFFAOYSA-N 0.000 description 2
- ZHQWPWQNVRCXAX-XQQFMLRXSA-N Val-Leu-Pro Chemical compound CC(C)C[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](C(C)C)N ZHQWPWQNVRCXAX-XQQFMLRXSA-N 0.000 description 2
- 210000000349 chromosome Anatomy 0.000 description 2
- 230000009089 cytolysis Effects 0.000 description 2
- OPTASPLRGRRNAP-UHFFFAOYSA-N cytosine Chemical compound NC=1C=CNC(=O)N=1 OPTASPLRGRRNAP-UHFFFAOYSA-N 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 238000004520 electroporation Methods 0.000 description 2
- 108010048367 enhanced green fluorescent protein Proteins 0.000 description 2
- 229940088598 enzyme Drugs 0.000 description 2
- DBLXOVFQHHSKRC-UHFFFAOYSA-N ethanesulfonic acid;2-piperazin-1-ylethanol Chemical compound CCS(O)(=O)=O.OCCN1CCNCC1 DBLXOVFQHHSKRC-UHFFFAOYSA-N 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 229940031154 kluyveromyces marxianus Drugs 0.000 description 2
- 239000006166 lysate Substances 0.000 description 2
- 230000035772 mutation Effects 0.000 description 2
- 108091027963 non-coding RNA Proteins 0.000 description 2
- 102000042567 non-coding RNA Human genes 0.000 description 2
- 150000003833 nucleoside derivatives Chemical class 0.000 description 2
- 244000052769 pathogen Species 0.000 description 2
- 230000001717 pathogenic effect Effects 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 108060006633 protein kinase Proteins 0.000 description 2
- 210000003370 receptor cell Anatomy 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 125000006850 spacer group Chemical group 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 231100000331 toxic Toxicity 0.000 description 2
- 230000002588 toxic effect Effects 0.000 description 2
- 230000035897 transcription Effects 0.000 description 2
- 238000013518 transcription Methods 0.000 description 2
- 230000032258 transport Effects 0.000 description 2
- UNXRWKVEANCORM-UHFFFAOYSA-N triphosphoric acid Chemical compound OP(O)(=O)OP(O)(=O)OP(O)(O)=O UNXRWKVEANCORM-UHFFFAOYSA-N 0.000 description 2
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 description 1
- 108020003589 5' Untranslated Regions Proteins 0.000 description 1
- ZKHQWZAMYRWXGA-KQYNXXCUSA-J ATP(4-) Chemical group C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O)[C@@H](O)[C@H]1O ZKHQWZAMYRWXGA-KQYNXXCUSA-J 0.000 description 1
- PWYFCPCBOYMOGB-LKTVYLICSA-N Ala-Gln-Trp Chemical compound C[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](CC1=CNC2=CC=CC=C21)C(=O)O)N PWYFCPCBOYMOGB-LKTVYLICSA-N 0.000 description 1
- 241000219195 Arabidopsis thaliana Species 0.000 description 1
- OSASDIVHOSJVII-WDSKDSINSA-N Arg-Cys Chemical compound SC[C@@H](C(O)=O)NC(=O)[C@@H](N)CCCNC(N)=N OSASDIVHOSJVII-WDSKDSINSA-N 0.000 description 1
- 239000004475 Arginine Substances 0.000 description 1
- QXHVOUSPVAWEMX-ZLUOBGJFSA-N Asp-Asp-Ser Chemical compound OC(=O)C[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CO)C(O)=O QXHVOUSPVAWEMX-ZLUOBGJFSA-N 0.000 description 1
- VILLWIDTHYPSLC-PEFMBERDSA-N Asp-Glu-Ile Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O VILLWIDTHYPSLC-PEFMBERDSA-N 0.000 description 1
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 description 1
- 241000726103 Atta Species 0.000 description 1
- YHZQPWIAFHYDJG-UHFFFAOYSA-M C(C)S(=O)(=O)[O-].OCCN1CCNCC1.[K+] Chemical compound C(C)S(=O)(=O)[O-].OCCN1CCNCC1.[K+] YHZQPWIAFHYDJG-UHFFFAOYSA-M 0.000 description 1
- 150000008574 D-amino acids Chemical class 0.000 description 1
- 230000006820 DNA synthesis Effects 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 101900264058 Escherichia coli Beta-galactosidase Proteins 0.000 description 1
- 108091029865 Exogenous DNA Proteins 0.000 description 1
- 108091081406 G-quadruplex Proteins 0.000 description 1
- MWERYIXRDZDXOA-QEWYBTABSA-N Gln-Ile-Phe Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O MWERYIXRDZDXOA-QEWYBTABSA-N 0.000 description 1
- ZOXBSICWUDAOHX-GUBZILKMSA-N Glu-Asn-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@@H](N)CCC(O)=O ZOXBSICWUDAOHX-GUBZILKMSA-N 0.000 description 1
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 1
- DTPOVRRYXPJJAZ-FJXKBIBVSA-N Gly-Arg-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)CN)CCCN=C(N)N DTPOVRRYXPJJAZ-FJXKBIBVSA-N 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- XKMLYUALXHKNFT-UUOKFMHZSA-N Guanosine-5'-triphosphate Chemical compound C1=2NC(N)=NC(=O)C=2N=CN1[C@@H]1O[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)[C@H]1O XKMLYUALXHKNFT-UUOKFMHZSA-N 0.000 description 1
- 101000595467 Homo sapiens T-complex protein 1 subunit gamma Proteins 0.000 description 1
- COWHUQXTSYTKQC-RWRJDSDZSA-N Ile-Thr-Glu Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCC(=O)O)C(=O)O)N COWHUQXTSYTKQC-RWRJDSDZSA-N 0.000 description 1
- 108020004684 Internal Ribosome Entry Sites Proteins 0.000 description 1
- 241000235650 Kluyveromyces marxianus Species 0.000 description 1
- 241000235058 Komagataella pastoris Species 0.000 description 1
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 1
- ONIBWKKTOPOVIA-BYPYZUCNSA-N L-Proline Chemical compound OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 0.000 description 1
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 1
- 150000008575 L-amino acids Chemical class 0.000 description 1
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 0.000 description 1
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 1
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 1
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 1
- HNDVDQJCIGZPNO-YFKPBYRVSA-N L-histidine Chemical compound OC(=O)[C@@H](N)CC1=CN=CN1 HNDVDQJCIGZPNO-YFKPBYRVSA-N 0.000 description 1
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 description 1
- LHSGPCFBGJHPCY-UHFFFAOYSA-N L-leucine-L-tyrosine Natural products CC(C)CC(N)C(=O)NC(C(O)=O)CC1=CC=C(O)C=C1 LHSGPCFBGJHPCY-UHFFFAOYSA-N 0.000 description 1
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 description 1
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 1
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 1
- AYFVYJQAPQTCCC-GBXIJSLDSA-N L-threonine Chemical compound C[C@@H](O)[C@H](N)C(O)=O AYFVYJQAPQTCCC-GBXIJSLDSA-N 0.000 description 1
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 1
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 1
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 description 1
- 239000012880 LB liquid culture medium Substances 0.000 description 1
- YOKVEHGYYQEQOP-QWRGUYRKSA-N Leu-Leu-Gly Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)NCC(O)=O YOKVEHGYYQEQOP-QWRGUYRKSA-N 0.000 description 1
- VDIARPPNADFEAV-WEDXCCLWSA-N Leu-Thr-Gly Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(O)=O VDIARPPNADFEAV-WEDXCCLWSA-N 0.000 description 1
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 1
- UQJOKDAYFULYIX-AVGNSLFASA-N Lys-Pro-Pro Chemical compound NCCCC[C@H](N)C(=O)N1CCC[C@H]1C(=O)N1[C@H](C(O)=O)CCC1 UQJOKDAYFULYIX-AVGNSLFASA-N 0.000 description 1
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- 108010052285 Membrane Proteins Proteins 0.000 description 1
- 241001529936 Murinae Species 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 239000008118 PEG 6000 Substances 0.000 description 1
- 229920000604 Polyethylene Glycol 200 Polymers 0.000 description 1
- 229920002560 Polyethylene Glycol 3000 Polymers 0.000 description 1
- 229920002562 Polyethylene Glycol 3350 Polymers 0.000 description 1
- 229920002584 Polyethylene Glycol 6000 Polymers 0.000 description 1
- 229920002594 Polyethylene Glycol 8000 Polymers 0.000 description 1
- LGMBKOAPPTYKLC-JYJNAYRXSA-N Pro-Phe-Arg Chemical compound C([C@@H](C(=O)N[C@@H](CCCNC(=N)N)C(O)=O)NC(=O)[C@H]1NCCC1)C1=CC=CC=C1 LGMBKOAPPTYKLC-JYJNAYRXSA-N 0.000 description 1
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 description 1
- 108091034057 RNA (poly(A)) Proteins 0.000 description 1
- 108700008625 Reporter Genes Proteins 0.000 description 1
- OJPHFSOMBZKQKQ-GUBZILKMSA-N Ser-Gln-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](N)CO OJPHFSOMBZKQKQ-GUBZILKMSA-N 0.000 description 1
- YQQKYAZABFEYAF-FXQIFTODSA-N Ser-Glu-Gln Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(N)=O)C(O)=O YQQKYAZABFEYAF-FXQIFTODSA-N 0.000 description 1
- YMTLKLXDFCSCNX-BYPYZUCNSA-N Ser-Gly-Gly Chemical compound OC[C@H](N)C(=O)NCC(=O)NCC(O)=O YMTLKLXDFCSCNX-BYPYZUCNSA-N 0.000 description 1
- DLPXTCTVNDTYGJ-JBDRJPRFSA-N Ser-Ile-Cys Chemical compound OC[C@H](N)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CS)C(O)=O DLPXTCTVNDTYGJ-JBDRJPRFSA-N 0.000 description 1
- HEYZPTCCEIWHRO-IHRRRGAJSA-N Ser-Met-Phe Chemical compound OC[C@H](N)C(=O)N[C@@H](CCSC)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 HEYZPTCCEIWHRO-IHRRRGAJSA-N 0.000 description 1
- WUXCHQZLUHBSDJ-LKXGYXEUSA-N Ser-Thr-Asp Chemical compound OC[C@H](N)C(=O)N[C@@H]([C@H](O)C)C(=O)N[C@@H](CC(O)=O)C(O)=O WUXCHQZLUHBSDJ-LKXGYXEUSA-N 0.000 description 1
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 1
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 1
- 241000187747 Streptomyces Species 0.000 description 1
- 102100036049 T-complex protein 1 subunit gamma Human genes 0.000 description 1
- CRZNCABIJLRFKZ-IUKAMOBKSA-N Thr-Ile-Asp Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)O)NC(=O)[C@H]([C@@H](C)O)N CRZNCABIJLRFKZ-IUKAMOBKSA-N 0.000 description 1
- WPSKTVVMQCXPRO-BWBBJGPYSA-N Thr-Ser-Ser Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(O)=O WPSKTVVMQCXPRO-BWBBJGPYSA-N 0.000 description 1
- 108091036066 Three prime untranslated region Proteins 0.000 description 1
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 description 1
- 239000004473 Threonine Substances 0.000 description 1
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 1
- FDKDGFGTHGJKNV-FHWLQOOXSA-N Tyr-Phe-Gln Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)O)NC(=O)[C@H](CC2=CC=C(C=C2)O)N FDKDGFGTHGJKNV-FHWLQOOXSA-N 0.000 description 1
- DDRBQONWVBDQOY-GUBZILKMSA-N Val-Ala-Arg Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCN=C(N)N)C(O)=O DDRBQONWVBDQOY-GUBZILKMSA-N 0.000 description 1
- XPKCFQZDQGVJCX-RHYQMDGZSA-N Val-Lys-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](C(C)C)N)O XPKCFQZDQGVJCX-RHYQMDGZSA-N 0.000 description 1
- VIKZGAUAKQZDOF-NRPADANISA-N Val-Ser-Glu Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CCC(O)=O VIKZGAUAKQZDOF-NRPADANISA-N 0.000 description 1
- PMKQKNBISAOSRI-XHSDSOJGSA-N Val-Tyr-Pro Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)N2CCC[C@@H]2C(=O)O)N PMKQKNBISAOSRI-XHSDSOJGSA-N 0.000 description 1
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 description 1
- 235000004279 alanine Nutrition 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 239000003125 aqueous solvent Substances 0.000 description 1
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
- 235000009582 asparagine Nutrition 0.000 description 1
- 229960001230 asparagine Drugs 0.000 description 1
- 235000003704 aspartic acid Nutrition 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 description 1
- 238000005842 biochemical reaction Methods 0.000 description 1
- 230000031018 biological processes and functions Effects 0.000 description 1
- 239000001506 calcium phosphate Substances 0.000 description 1
- 229910000389 calcium phosphate Inorganic materials 0.000 description 1
- 235000011010 calcium phosphates Nutrition 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 238000000975 co-precipitation Methods 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- NKLPQNGYXWVELD-UHFFFAOYSA-M coomassie brilliant blue Chemical group [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=C1 NKLPQNGYXWVELD-UHFFFAOYSA-M 0.000 description 1
- 239000000287 crude extract Substances 0.000 description 1
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 1
- 235000018417 cysteine Nutrition 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 229940104302 cytosine Drugs 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- MTHSVFCYNBDYFN-UHFFFAOYSA-N diethylene glycol Chemical compound OCCOCCO MTHSVFCYNBDYFN-UHFFFAOYSA-N 0.000 description 1
- 238000007877 drug screening Methods 0.000 description 1
- 230000001516 effect on protein Effects 0.000 description 1
- 238000005538 encapsulation Methods 0.000 description 1
- 239000003623 enhancer Substances 0.000 description 1
- 238000012869 ethanol precipitation Methods 0.000 description 1
- 239000013604 expression vector Substances 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 238000010363 gene targeting Methods 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 210000004602 germ cell Anatomy 0.000 description 1
- 235000013922 glutamic acid Nutrition 0.000 description 1
- 239000004220 glutamic acid Substances 0.000 description 1
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 1
- 108010078144 glutaminyl-glycine Proteins 0.000 description 1
- 108010049041 glutamylalanine Proteins 0.000 description 1
- 108010010147 glycylglutamine Proteins 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 1
- 210000005260 human cell Anatomy 0.000 description 1
- 230000003053 immunization Effects 0.000 description 1
- 238000002649 immunization Methods 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 description 1
- 229960000310 isoleucine Drugs 0.000 description 1
- 108010012058 leucyltyrosine Proteins 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 210000001161 mammalian embryo Anatomy 0.000 description 1
- 238000010297 mechanical methods and process Methods 0.000 description 1
- 229930182817 methionine Natural products 0.000 description 1
- 238000000520 microinjection Methods 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 230000004481 post-translational protein modification Effects 0.000 description 1
- 229960004109 potassium acetate Drugs 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 108010053725 prolylvaline Proteins 0.000 description 1
- 230000012846 protein folding Effects 0.000 description 1
- 230000003161 proteinsynthetic effect Effects 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 238000003259 recombinant expression Methods 0.000 description 1
- 238000010188 recombinant method Methods 0.000 description 1
- 239000003161 ribonuclease inhibitor Substances 0.000 description 1
- 210000003705 ribosome Anatomy 0.000 description 1
- 229920002477 rna polymer Polymers 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 108010026333 seryl-proline Proteins 0.000 description 1
- 239000013605 shuttle vector Substances 0.000 description 1
- 230000011664 signaling Effects 0.000 description 1
- 238000002791 soaking Methods 0.000 description 1
- 239000001632 sodium acetate Substances 0.000 description 1
- 235000017281 sodium acetate Nutrition 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 238000003151 transfection method Methods 0.000 description 1
- 230000014621 translational initiation Effects 0.000 description 1
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
- 241001515965 unidentified phage Species 0.000 description 1
- 229940035893 uracil Drugs 0.000 description 1
- 239000004474 valine Substances 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 230000009614 wildtype growth Effects 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
-
- 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
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/62—DNA sequences coding for fusion proteins
- C12N15/625—DNA sequences coding for fusion proteins containing a sequence coding for a signal sequence
-
- 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
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
- C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/60—Fusion polypeptide containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP]
Landscapes
- Genetics & Genomics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Physics & Mathematics (AREA)
- Microbiology (AREA)
- Plant Pathology (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention provides a signal peptide related sequence and application thereof in protein synthesis, in particular to a signal peptide with the effect of improving protein expression and a coding sequence thereof, and a nucleic acid construct formed by operably connecting the coding sequence of the signal peptide and the coding sequence of a foreign protein can obviously improve the efficiency of foreign protein synthesis and simplify the expression and purification processes of a target foreign protein. Meanwhile, the invention provides a vector or a vector combination, a genetic engineering cell and a kit corresponding to the protein, so that the protein can be applied to protein synthesis.
Description
Technical Field
The invention relates to the field of biotechnology, in particular to a signal peptide related sequence and application thereof in protein synthesis.
Background
Proteins are important molecules in cells, and are involved in performing almost all functions of cells. The difference in the sequence and structure of the protein determines the difference in its function (1, 2). Within the cell, proteins can catalyze various biochemical reactions as enzymes, can coordinate various activities of the organism as signaling molecules, can support biological morphology, store energy, transport molecules, and mobilize the organism (2). In the biomedical field, protein antibodies are important means for treating diseases such as cancer as targeted drugs (2).
Signal peptides are short peptides at the N-terminus of proteins that carry the secretory information of proteins and are widely distributed in all prokaryotes and eukaryotes (3, 4). The research on signal peptides has been focused on many scientific and industrial fields, including the production of recombinant proteins, disease diagnosis, immunization and many biological experimental techniques (4, 5). Many studies have shown that signal peptides play a very important role in recombinant protein production (6, 7). However, some of the functions of signal peptides in protein expression and transmembrane structure formation remain elusive (4,8, 9).
In addition to the understanding of intracellular protein synthesis, protein synthesis can also be carried out extracellularly. The in vitro protein synthesis system is generally characterized in that components such as mRNA or DNA template, RNA polymerase, amino acid, ATP and the like are added into a lysis system of bacteria, fungi, plant cells or animal cells to complete the rapid and efficient translation of foreign proteins (10, 11). Currently, commercial in vitro protein expression systems that are frequently tested include the e.coli system (ECE), Rabbit Reticulocyte Lysate (RRL), Wheat Germ (WGE), Insect Cell Extract (ICE) and human systems (11, 12). Compared with the traditional in vivo recombinant expression system, the in vitro cell-free protein synthesis system has multiple advantages, such as the capability of expressing special proteins which have toxic action on cells or contain unnatural amino acids (such as D-amino acids), the capability of directly taking PCR products as templates to simultaneously synthesize multiple proteins in parallel and the development of high-throughput drug screening and proteomics research (10-12).
Studies have shown that part of the signal peptide sequence has some promotion effect on protein expression, while DNA templates used in vitro synthesis usually do not have signal peptide related sequences (13). Therefore, in an in vitro protein synthesis system, a short polypeptide sequence with the length of less than 30 amino acids is generally inserted into the N-terminal of the target protein to improve the translation efficiency of the target protein, but the insertion of some short peptides can significantly influence the structure and function of the target protein (4, 14).
Therefore, there is an urgent need in the art to provide a signal peptide-related sequence that can be applied to an in vitro protein expression system, and can significantly improve the yield of a target protein, reduce the cost of protein expression, and improve the protein translation efficiency.
Disclosure of Invention
The invention aims to provide a signal peptide related sequence which can be applied to an in vitro protein expression system, can obviously improve the yield of target protein, reduce the cost of protein expression and improve the protein translation efficiency.
In a first aspect of the invention, there is provided a nucleic acid construct comprising a first nucleotide sequence encoding a signal peptide operably linked to a second nucleotide sequence encoding a foreign protein, the 3' end of the first nucleotide sequence being upstream of the second nucleotide sequence, and the first nucleotide sequence being selected from the group consisting of:
(a) a nucleotide sequence encoding any one of the following signal peptides: the amino acid sequence is SEQ ID NO: 14-24;
(b) SEQ ID NO: 1-13.
In another preferred embodiment, the nucleic acid construct has a structure of formula I from 5 'to 3':
Z1-Z2-Z3 (I)
in the formula (I), the compound is shown in the specification,
Z1-Z3 are each an element used to construct the construct;
each "-" is independently a bond or a nucleotide linking sequence;
z1 is the coding sequence of signal peptide;
z2 is nothing or a linking sequence;
z3 is the coding sequence of no or exogenous protein;
wherein the coding sequence of the signal peptide is selected from the group consisting of:
(a) a polynucleotide encoding a polypeptide as set forth in SEQ ID No. 14-24;
(b) a polynucleotide having a sequence as set forth in any one of SEQ ID No. 1-13;
(c) a polynucleotide having a nucleotide sequence homology of 75% or more (preferably 85% or more, more preferably 90% or more or 95% or more or 98% or more or 99%) with any one of the sequences shown in SEQ ID No. 1-13;
(d) 1-13, or a polynucleotide in which 1-60 (preferably 1-30, more preferably 1-10) nucleotides are truncated or added at the 5 'end and/or 3' end of the polynucleotide shown in SEQ ID NO;
(e) a polynucleotide complementary to any one of the polynucleotides of (a) - (d).
In another preferred embodiment, the nucleic acid construct has a structure of formula II from 5 'to 3':
Z1-Z2-Z3 (II)
in the formula (I), the compound is shown in the specification,
Z1-Z3 are each an element used to construct the construct;
each "-" is independently a bond or a nucleotide linking sequence;
z1 is the coding sequence of signal peptide;
z2 is a linker sequence;
z3 is the coding sequence of no or exogenous protein;
wherein the coding sequence of the signal peptide is selected from the group consisting of:
(a) a polynucleotide encoding a polypeptide as set forth in SEQ ID No. 14-24;
(b) a polynucleotide having a sequence as set forth in any one of SEQ ID No. 1-13;
(c) a polynucleotide having a nucleotide sequence homology of 75% or more (preferably 85% or more, more preferably 90% or more or 95% or more or 98% or more or 99%) with any one of the sequences shown in SEQ ID No. 1-13;
(d) 1-13, or a polynucleotide in which 1-60 (preferably 1-30, more preferably 1-10) nucleotides are truncated or added at the 5 'end and/or 3' end of the polynucleotide shown in SEQ ID NO;
(e) a polynucleotide complementary to any one of the polynucleotides of (a) - (d).
In another preferred embodiment, the nucleic acid construct has a structure of formula III from 5 'to 3':
Z1-Z2-Z3 (III)
in the formula (I), the compound is shown in the specification,
Z1-Z3 are each an element used to construct the construct;
each "-" is independently a bond or a nucleotide linking sequence;
z1 is the coding sequence of signal peptide;
z2 is a linker sequence;
z3 is the coding sequence of the foreign protein;
wherein the coding sequence of the signal peptide is selected from the group consisting of:
(a) a polynucleotide encoding a polypeptide as set forth in SEQ ID No. 14-24;
(b) a polynucleotide having a sequence as set forth in any one of SEQ ID No. 1-13;
(c) a polynucleotide having a nucleotide sequence homology of 75% or more (preferably 85% or more, more preferably 90% or more or 95% or more or 98% or more or 99%) with any one of the sequences shown in SEQ ID No. 1-13;
(d) 1-13, or a polynucleotide in which 1-60 (preferably 1-30, more preferably 1-10) nucleotides are truncated or added at the 5 'end and/or 3' end of the polynucleotide shown in SEQ ID NO;
(e) a polynucleotide complementary to any one of the polynucleotides of (a) - (d).
In another preferred embodiment, the operably linked is directly linked or linked through a linking sequence.
In another preferred embodiment, the linker sequence is SEQ ID NO: 25.
In another preferred embodiment, the amino acid sequence of the linker sequence is as shown in SEQ ID NO. 26.
In another preferred embodiment, the coding sequence of the signal peptide is a codon optimized coding sequence.
In another preferred embodiment, the coding sequence of the signal peptide is as shown in SEQ ID NO. 1-13.
In another preferred embodiment, the amino acid sequence of the signal peptide has the sequence shown in SEQ ID NO. 14-24 or an active fragment thereof, or has a homology of more than or equal to 85%, preferably more than or equal to 90% with the amino acid sequence shown in SEQ ID NO. 14-24; more preferably 95% homology or more; most preferably, homology of more than or equal to 97%, such as more than 98%, more than 99%) and has the same activity as the sequence shown in SEQ ID NO. 14-24.
In another preferred embodiment, the coding sequence of the signal peptide is shown in SEQ ID NO. 11-13.
In another preferred embodiment, the coding sequence of the signal peptide is as shown in SEQ ID NO. 2-7.
In another preferred embodiment, the coding sequence of the signal peptide is shown in SEQ ID No. 1.
In another preferred embodiment, the coding sequence of the signal peptide is shown in SEQ ID NO. 8-10.
In another preferred embodiment, the amino acid sequence of the signal peptide is shown in SEQ ID NO. 22-24.
In another preferred embodiment, the amino acid sequence of the signal peptide is shown in SEQ ID NO. 15-20.
In another preferred embodiment, the amino acid sequence of the signal peptide is shown in SEQ ID No. 14.
In another preferred embodiment, the amino acid sequence of the signal peptide is shown in SEQ ID No. 21.
In another preferred embodiment, the linker sequence is a codon optimized linker sequence.
In another preferred embodiment, the linker sequence has a sequence that is not prone to secondary structure formation (e.g., AT-rich sequence, hairpin-free structure, G-quadruplex-free (G-quadruplex), etc.), and is not enriched in rare codons.
In another preferred embodiment, the linker sequence is selected from the group consisting of;
(i) a polynucleotide having a sequence as set forth in SEQ ID No. 25;
(ii) polynucleotide having a nucleotide sequence homology of 75% or more (preferably 85% or more, more preferably 90% or 95% or 98% or 99% or more) with the sequence shown in SEQ ID No. 25;
(iii) a polynucleotide in which 1 to 60 (preferably 1 to 30, more preferably 1 to 10) nucleotides are truncated or added at the 5 'end and/or the 3' end of the polynucleotide shown in SEQ ID No. 25;
(iv) (iv) a polynucleotide complementary to any one of the polynucleotides of (i) to (iii).
In another preferred embodiment, the foreign protein is from a prokaryote or a eukaryote.
In another preferred embodiment, the foreign protein is from an animal, plant, pathogen.
In another preferred embodiment, the foreign protein is from a mammal, preferably a primate, a rodent, including a human, a mouse, a rat.
In another preferred embodiment, the foreign protein is selected from the group consisting of: luciferin protein, luciferase (e.g., firefly luciferase), green fluorescent protein, yellow fluorescent protein, aminoacyl tRNA synthetase, glyceraldehyde-3-phosphate dehydrogenase, catalase, actin, variable region of an antibody, luciferase mutation, alpha-amylase, enteromycin A, hepatitis C virus E2 glycoprotein, insulin precursor, interferon alpha A, interleukin-1 beta, lysozyme, serum albumin, single chain antibody fragment (scFV), transthyretin, tyrosinase, xylanase, or a combination thereof.
In another preferred embodiment, the coding sequence for the foreign protein encodes a protein selected from the group consisting of: a luciferin protein, or a luciferase (such as firefly luciferase), a green fluorescent protein, a yellow fluorescent protein, an aminoacyl tRNA synthetase, a glyceraldehyde-3-phosphate dehydrogenase, a catalase, an actin, an antibody or variable region thereof, a luciferase mutant, or a combination thereof.
In another preferred embodiment, the nucleic acid construct further comprises a promoter upstream of the 5' end.
In another preferred embodiment, the promoter comprises a constitutive or inducible promoter.
In another preferred embodiment, the promoter is selected from the group consisting of: a T7 promoter, a T3 promoter, an SP6 promoter, or a combination thereof.
In another preferred embodiment, the nucleic acid construct further comprises an enhancer element, an RBS ribosome binding sequence, a Spacer sequence (Spacer), other related sequences for RNA transcription, translation, or a combination thereof.
In another preferred embodiment, the enhancer element comprises an internal ribosome entry site element (IRES), a ribosome binding site element, a non-coding sequence, or a combination thereof.
In another preferred embodiment, the IRES element is derived from one or more cells selected from the group consisting of: prokaryotic cells and eukaryotic cells.
In another preferred embodiment, the eukaryotic cells include higher eukaryotic cells.
In another preferred embodiment, the IRES element comprises an endogenous IRES element and an exogenous IRES element.
In another preferred embodiment, the IRES element is derived from one or more cells selected from the group consisting of: human (human), Chinese hamster ovary Cells (CHO), insect cells (instect), Wheat germ (Wheat cells), Rabbit reticulocyte (Rabbit reticulocyte).
In another preferred embodiment, the IRES element is selected from the group consisting of: ScGPR1, ScFLO8, ScNCE102, ScMSN1, KlFLO8, KlNCE102, KlMSN1, GAA, Omega10A, or a combination thereof.
In a second aspect of the invention, there is provided a signal peptide whose amino acid sequence is encoded by the first nucleotide sequence of the first aspect.
In another preferred embodiment, the amino acid sequence of the signal peptide is shown in any one of SEQ ID No. 14-24.
In a third aspect of the invention, there is provided a vector or combination of vectors comprising a nucleic acid construct according to the first aspect of the invention.
In a fourth aspect of the invention, there is provided a genetically engineered cell having a nucleic acid construct according to the first aspect of the invention integrated at one or more sites in its genome or comprising a vector or combination of vectors according to the third aspect of the invention.
In another preferred embodiment, the genetically engineered cell comprises a prokaryotic cell and a eukaryotic cell.
In another preferred embodiment, the eukaryotic cells include higher eukaryotic cells.
In another preferred embodiment, the genetically engineered cell is selected from the group consisting of: human cells (e.g., Hela cells), chinese hamster ovary cells, insect cells, wheat germ cells, rabbit reticulocyte, yeast cells, or combinations thereof.
In another preferred embodiment, the genetically engineered cell is a yeast cell.
In another preferred embodiment, the yeast cell is selected from the group consisting of: saccharomyces cerevisiae, Kluyveromyces yeast, or a combination thereof.
In another preferred embodiment, the yeast of the genus kluyveromyces is selected from the group consisting of: kluyveromyces lactis, Kluyveromyces marxianus, Kluyveromyces multibuyveri, or a combination thereof.
In a fifth aspect of the invention, a kit is provided, wherein the kit comprises reagents selected from one or more of the following groups:
(a) a nucleic acid construct according to the first aspect of the invention;
(b) a vector or combination of vectors according to the third aspect of the invention; and
(c) the genetically engineered cell according to the fourth aspect of the invention.
In another preferred embodiment, the kit further comprises (d) a eukaryotic in vitro biosynthesis system (e.g., a eukaryotic in vitro protein synthesis system).
In another preferred embodiment, the eukaryotic in vitro biosynthetic system is selected from the group consisting of: a yeast in vitro biosynthesis system, a chinese hamster ovary cell in vitro biosynthesis system, an insect cell in vitro biosynthesis system, a Hela cell in vitro biosynthesis system, or a combination thereof.
In another preferred embodiment, the eukaryotic in vitro biosynthetic system comprises a eukaryotic in vitro protein synthetic system.
In another preferred embodiment, the eukaryotic in vitro protein synthesis system is selected from the group consisting of: a yeast in vitro protein synthesis system, a chinese hamster ovary cell in vitro protein synthesis system, an insect cell in vitro protein synthesis system, a Hela cell in vitro protein synthesis system, or a combination thereof.
In another preferred embodiment, the yeast in vitro biosynthesis system (e.g., yeast in vitro protein synthesis system) is a Kluyveromyces in vitro biosynthesis system (e.g., Kluyveromyces in vitro protein synthesis system), preferably a Kluyveromyces lactis in vitro biosynthesis system (e.g., Kluyveromyces lactis in vitro protein synthesis system).
In another preferred embodiment, the yeast in vitro biosynthesis system is a Kluyveromyces in vitro biosynthesis system.
In a sixth aspect, the invention provides the use of a nucleic acid construct according to the first aspect, a signal peptide according to the second aspect, a vector or combination of vectors according to the third aspect, a genetically engineered cell according to the fourth aspect or a kit according to the fifth aspect in an in vitro protein synthesis system.
In a seventh aspect of the present invention, there is provided an in vitro protein synthesis method, comprising the steps of:
(i) providing an in vitro biosynthetic system comprising a nucleic acid construct according to the first aspect of the invention;
(ii) (ii) incubating the in vitro biosynthetic system of step (i) under suitable conditions for a reaction time to synthesize the foreign protein.
In another preferred embodiment, the in vitro protein synthesis method further comprises (iii) isolating or detecting said foreign protein, optionally from said eukaryotic in vitro biosynthesis system.
In another preferred embodiment, in the step (ii), the reaction temperature is 20 to 37 ℃, preferably 22 to 35 ℃.
In another preferred embodiment, in the step (ii), the reaction time is 1 to 72 hours, preferably 2 to 24 hours.
In another preferred embodiment, the in vitro biosynthesis system may be selected from a yeast in vitro biosynthesis system (e.g., a yeast in vitro protein synthesis system).
In another preferred embodiment, the yeast in vitro biosynthesis system (e.g., yeast in vitro protein synthesis system) is a Kluyveromyces in vitro biosynthesis system (e.g., Kluyveromyces in vitro protein synthesis system) (preferably a Kluyveromyces lactis in vitro biosynthesis system, e.g., Kluyveromyces lactis in vitro protein synthesis system).
In another preferred embodiment, the foreign protein is from a prokaryote or a eukaryote.
In another preferred embodiment, the foreign protein is from an animal, plant, pathogen.
In another preferred embodiment, the foreign protein is from a mammal, preferably a primate, a rodent, including a human, a mouse, a rat.
In another preferred embodiment, the foreign protein is selected from the group consisting of: luciferin protein, or luciferase (e.g., firefly luciferase), green fluorescent protein, yellow fluorescent protein, aminoacyl tRNA synthetase, glyceraldehyde-3-phosphate dehydrogenase, catalase, actin, variable region of an antibody, luciferase mutation, alpha-amylase, enteromycin A, hepatitis C virus E2 glycoprotein, insulin precursor, interferon alpha A, interleukin-1 beta, lysozyme, serum albumin, single chain antibody fragment (scFV), transthyretin, tyrosinase, xylanase, or a combination thereof.
In another preferred embodiment, the coding sequence of the foreign protein encodes a foreign protein selected from the group consisting of: luciferin protein, luciferase (e.g., firefly luciferase), green fluorescent protein, yellow fluorescent protein, aminoacyl tRNA synthetase, glyceraldehyde-3-phosphate dehydrogenase, catalase, actin, variable region of an antibody, luciferase mutant, alpha-amylase, enteromycin A, hepatitis C virus E2 glycoprotein, insulin precursor, interferon alpha A, interleukin-1 beta, lysozyme, serum albumin, single chain antibody fragment (scFV), transthyretin, tyrosinase, xylanase, or a combination thereof.
In an eighth aspect, the invention provides an isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide encoding a polypeptide as set forth in SEQ ID No. 14-24;
(b) a polynucleotide having a sequence as set forth in any one of SEQ ID No. 1-13;
(c) a polynucleotide having a nucleotide sequence homology of 75% or more (preferably 85% or more, more preferably 90% or more or 95% or more or 98% or more or 99%) with any one of the sequences shown in SEQ ID No. 1-13;
(d) 1-13, or a polynucleotide in which 1-60 (preferably 1-30, more preferably 1-10) nucleotides are truncated or added at the 5 'end and/or 3' end of the polynucleotide shown in SEQ ID NO;
(e) a polynucleotide complementary to any one of the polynucleotides of (a) - (d).
In another preferred embodiment, the polynucleotide is a nucleotide sequence encoding a signal peptide.
In another preferred embodiment, the polynucleotide comprises a DNA sequence.
In a ninth aspect, the invention provides a linker sequence selected from the group consisting of:
(i) a polynucleotide having a sequence as set forth in SEQ ID No. 25;
(ii) polynucleotide having a nucleotide sequence homology of 75% or more (preferably 85% or more, more preferably 90% or 95% or 98% or 99% or more) with the sequence shown in SEQ ID No. 25;
(iii) a polynucleotide in which 1 to 60 (preferably 1 to 30, more preferably 1 to 10) nucleotides are truncated or added at the 5 'end and/or the 3' end of the polynucleotide shown in SEQ ID No. 25;
(iv) (iv) a polynucleotide complementary to any one of the polynucleotides of (i) to (iii).
It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.
Drawings
FIG. 1 shows the basic biological process from DNA to protein.
FIG. 2 shows Relative Fluorescence unit values (RFUs) of Enhanced green Fluorescence protein (eGFP) synthesized by 13 related sequences of the signal peptide of the present invention in an in vitro protein synthesis system.
Detailed Description
The present inventors have made extensive and intensive studies and, as a result of extensive screening and investigation, have for the first time found a novel signal peptide for improving the protein translation efficiency of an in vitro protein synthesis system and a nucleic acid construct comprising a signal peptide-encoding sequence, which comprises a first nucleotide sequence (including a codon-optimized or non-optimized signal peptide-encoding sequence) encoding a signal peptide operably linked to a second nucleotide sequence encoding a foreign protein. Experiments show that the signal intensity of the synthesized foreign protein is remarkably improved (p is less than 0.05) compared with that of a control group by applying the nucleic acid construct or the signal peptide sequence in an in-vitro protein synthesis system (such as a yeast in-vitro protein synthesis system). The invention also simplifies the expression and purification mode of the foreign protein.
Protein synthesis system
Protein synthesis refers to the process by which an organism synthesizes a protein according to genetic information on messenger ribonucleic acid (mRNA) transcribed from deoxyribonucleic acid (DNA), as shown in fig. 1. Protein biosynthesis is also known as Translation (Translation), the process by which the sequence of bases in an mRNA molecule is converted into the sequence of amino acids in a protein or polypeptide chain. This is the second step in gene expression, the final stage in the production of the gene product protein. Different tissue cells have different physiological functions because they express different genes to produce proteins with special functions, and at least 200 components involved in protein biosynthesis are mainly composed of mRNA, tRNA, ribonucleate, related enzymes and protein factors.
The in vitro protein synthesis system is generally characterized in that mRNA or DNA template, RNA polymerase, amino acid, ATP and other components are added into a lysis system of bacteria, fungi, plant cells or animal cells to complete the rapid and efficient translation of exogenous protein. Currently, commercial in vitro protein expression systems that are frequently tested include the e.coli system (ECE), Rabbit Reticulocyte Lysate (RRL), Wheat Germ (WGE), Insect Cell Extract (ICE) and human-derived systems.
Yeast (yeast) combines the advantages of simple culture, efficient protein folding, and post-translational modification. Among them, Saccharomyces cerevisiae and Pichia pastoris are model organisms expressing complex eukaryotic proteins and membrane proteins, and yeast may be used as material for preparing in vitro translation systems.
Kluyveromyces (Kluyveromyces) is a species of ascosporogenous yeast, of which Kluyveromyces marxianus and Kluyveromyces lactis (Kluyveromyces lactis) are industrially widely used. In comparison with other yeasts, kluyveromyces lactis has many advantages such as superior secretion ability, better large-scale fermentation characteristics, a level of food safety, and the ability to modify proteins post-translationally.
In the present invention, one preferred protein synthesis system is an in vitro protein synthesis system. The in vitro protein synthesis system of the present invention is not particularly limited, and one preferred in vitro protein synthesis system is a Kluyveromyces expression system (more preferably, a Kluyveromyces lactis expression system).
In the present invention, the in vitro protein synthesis system comprises: yeast cell extract and optionally a solvent, which is water or an aqueous solvent.
In a particularly preferred embodiment, the in vitro protein synthesis system provided by the present invention further comprises: 4-hydroxyethyl piperazine ethanesulfonic acid, potassium acetate, magnesium acetate, nucleoside triphosphate mixtures, amino acid mixtures, phosphocreatine, Dithiothreitol (DTT), phosphocreatine kinase, RNase inhibitors, luciferin, luciferase DNA, RNA polymerase.
In the present invention, the RNA polymerase is not particularly limited and may be selected from one or more RNA polymerases, and a typical RNA polymerase is T7 RNA polymerase.
In the present invention, the proportion of the yeast cell extract in the in vitro protein synthesis system is not particularly limited, and usually the yeast cell extract accounts for 20 to 70%, preferably 30 to 60%, more preferably 40 to 50% of the in vitro protein synthesis system.
In the present invention, the yeast cell extract does not contain intact cells, and typical yeast cell extracts include ribosomes for protein translation, transfer RNAs, aminoacyl tRNA synthetases, initiation and elongation factors required for protein synthesis, and termination and release factors. In addition, the yeast extract also contains some other proteins, especially soluble proteins, which originate in the cytoplasm of the yeast cell.
In the present invention, the yeast cell extract contains 10-100mg/mL of protein, preferably 20-80 mg/mL. The method for determining the protein content is a Coomassie brilliant blue determination method.
In the present invention, the preparation method of the yeast cell extract is not limited, and a preferred preparation method comprises the steps of:
(i) providing a yeast cell;
(ii) washing the yeast cells to obtain washed yeast cells;
(iii) subjecting the washed yeast cells to cell disruption treatment, thereby obtaining a crude yeast extract;
(iv) and carrying out solid-liquid separation on the yeast crude extract to obtain a liquid part, namely the yeast cell extract.
In the present invention, the solid-liquid separation method is not particularly limited, but centrifugation is preferable.
In the present invention, the centrifugation conditions are not particularly limited, and the centrifugation conditions are 5000-.
In the present invention, the centrifugation time is not particularly limited, and the centrifugation time is 0.5min to 2h, preferably 20min to 50 min.
In the present invention, the temperature of the centrifugation is not particularly limited, and it is preferable that the centrifugation is performed at 1 to 10 ℃, preferably, 2 to 6 ℃.
In the present invention, the washing treatment is not particularly limited, and a preferable washing treatment is a treatment with a washing solution at a pH of 7 to 8 (preferably, 7.4), the washing solution is not particularly limited, and typically the washing solution is selected from the group consisting of: potassium 4-hydroxyethylpiperazine ethanesulfonate, potassium acetate, magnesium acetate, or a combination thereof.
In the present invention, the manner of the cell disruption treatment is not particularly limited, and preferred cell disruption treatments include high-pressure disruption, freeze-thaw (e.g., liquid nitrogen low-temperature) disruption.
The nucleoside triphosphate mixture in the in vitro protein synthesis system is adenosine triphosphate, guanosine triphosphate, cytosine nucleoside triphosphate and uracil nucleoside triphosphate. In the present invention, the concentration of each mononucleotide is not particularly limited, and usually the concentration of each mononucleotide is 0.5 to 5mM, preferably 1.0 to 2.0 mM.
The amino acid mixture in the in vitro protein synthesis system may comprise natural or unnatural amino acids, and may comprise D-or L-amino acids. Representative amino acids include (but are not limited to) the 20 natural amino acids: glycine, alanine, valine, leucine, isoleucine, phenylalanine, proline, tryptophan, serine, tyrosine, cysteine, methionine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine, and histidine. The concentration of each amino acid is usually 0.01-0.5mM, preferably 0.02-0.2mM, such as 0.05, 0.06, 0.07, 0.08 mM.
In a preferred embodiment, the in vitro protein synthesis system further comprises polyethylene glycol (PEG) or an analog thereof. The concentration of polyethylene glycol or an analog thereof is not particularly limited, and usually, the concentration (w/v) of polyethylene glycol or an analog thereof is 0.1 to 8%, preferably 0.5 to 4%, more preferably 1 to 2%, based on the total weight of the protein synthesis system. Representative PEG examples include (but are not limited to): PEG3000, PEG8000, PEG6000 and PEG 3350. It is understood that the systems of the present invention may also include other polyethylene glycols of various molecular weights (e.g., PEG200, 400, 1500, 2000, 4000, 6000, 8000, 10000, etc.).
In a preferred embodiment, the in vitro protein synthesis system further comprises sucrose. The concentration of sucrose is not particularly limited, and generally, the concentration of sucrose is 0.03 to 40 wt%, preferably 0.08 to 10 wt%, more preferably 0.1 to 5 wt%, based on the total weight of the protein synthesis system.
A particularly preferred in vitro protein synthesis system comprises, in addition to yeast extract, the following components: 22mM of 4-hydroxyethyl piperazine ethanesulfonic acid with the pH value of 7.4, 30-150mM of potassium acetate, 1.0-5.0mM of magnesium acetate, 1.5-4mM of nucleoside triphosphate mixture, 0.08-0.24mM of amino acid mixture, 25mM of creatine phosphate, 1.7mM of dithiothreitol, 0.27mg/mL of phosphocreatine kinase, 1% -4% of polyethylene glycol, 0.5% -2% of sucrose, 8-20 ng/mu L of DNA of firefly luciferase and 0.027-0.054mg/mL of T7 RNA polymerase.
Coding sequence of foreign protein (foreign DNA)
As used herein, the term "coding sequence for a foreign protein" is used interchangeably with "foreign DNA" and refers to a foreign DNA molecule used to direct protein synthesis. Typically, the DNA molecule is linear or circular. The DNA molecule contains a sequence encoding a foreign protein.
In the present invention, examples of the sequence encoding the foreign protein include (but are not limited to): genome sequence and cDNA sequence. The sequence for encoding the foreign protein also comprises a promoter sequence, a 5 'untranslated sequence and a 3' untranslated sequence.
In the present invention, the selection of the foreign DNA is not particularly limited, and in general, the foreign DNA encodes a protein selected from the group consisting of: luciferin protein, luciferase (e.g., firefly luciferase), green fluorescent protein, yellow fluorescent protein, aminoacyl tRNA synthetase, glyceraldehyde-3-phosphate dehydrogenase, catalase, actin, antibody or variable region thereof, luciferase mutant, or a combination thereof.
The foreign DNA may also encode a protein selected from the group consisting of: alpha-amylase, enteromycin A, hepatitis C virus E2 glycoprotein, insulin precursor, interferon alpha A, interleukin-1 beta, lysozyme, serum albumin, single chain antibody fragments (scFV), transthyretin, tyrosinase, xylanase, or a combination thereof.
In a preferred embodiment, the exogenous DNA encodes a protein selected from the group consisting of: green fluorescent protein (eGFP), Yellow Fluorescent Protein (YFP), escherichia coli β -galactosidase (lactasise, LacZ), human Lysine-tRNA synthetase (Lysine-tRNA synthetase), human Leucine-tRNA synthetase (Leucine-tRNA synthetase), arabidopsis thaliana Glyceraldehyde 3-phosphate dehydrogenase (Glyceraldehyde-3-phosphate dehydrogenase), murine Catalase (Catalase), or combinations thereof.
Nucleic acid constructs
In a first aspect, the present invention provides a nucleic acid construct comprising a first nucleotide sequence encoding a signal peptide operably linked to a second nucleotide sequence encoding a foreign protein, the 3' end of the first nucleotide sequence being upstream of the second nucleotide sequence, and the first nucleotide sequence being selected from the group consisting of:
(a) a nucleotide sequence encoding any one of the following signal peptides: the amino acid sequence is SEQ ID NO: 14-24;
(b) SEQ ID NO: 1-13.
The term "operably linked" refers to a functional spatial arrangement of two or more nucleotide regions or nucleotide sequences. For example: the nucleotide sequence encoding the signal peptide is placed at a specific position relative to the nucleotide sequence of the foreign protein, so that an effect of increasing the expression of the foreign protein is obtained. The operable linkage is direct linkage or linkage through a linking sequence.
In a preferred embodiment, the nucleic acid construct of the invention has a structure of formula I from 5 'to 3':
Z1-Z2-Z3 (I)
in the formula (I), the compound is shown in the specification,
Z1-Z3 are each an element used to construct the construct;
each "-" is independently a bond or a nucleotide linking sequence;
z1 is the coding sequence of signal peptide;
z2 is nothing or a linking sequence;
z3 is the coding sequence of no or exogenous protein;
wherein the coding sequence of the signal peptide is selected from the group consisting of:
(a) a polynucleotide encoding a polypeptide as set forth in SEQ ID No. 14-24;
(b) a polynucleotide having a sequence as set forth in any one of SEQ ID No. 1-13;
(c) a polynucleotide having a nucleotide sequence homology of 75% or more (preferably 85% or more, more preferably 90% or more or 95% or more or 98% or more or 99%) with any one of the sequences shown in SEQ ID No. 1-13;
(d) 1-13, or a polynucleotide in which 1-60 (preferably 1-30, more preferably 1-10) nucleotides are truncated or added at the 5 'end and/or 3' end of the polynucleotide shown in SEQ ID NO;
(e) a polynucleotide complementary to any one of the polynucleotides of (a) - (d).
In a preferred embodiment, the nucleic acid construct of the invention has a structure of formula II from 5 'to 3':
Z1-Z2-Z3 (II)
in the formula (I), the compound is shown in the specification,
Z1-Z3 are each an element used to construct the construct;
each "-" is independently a bond or a nucleotide linking sequence;
z1 is the coding sequence of signal peptide;
z2 is a linker sequence;
z3 is the coding sequence of no or exogenous protein;
wherein the coding sequence of the signal peptide is selected from the group consisting of:
(a) a polynucleotide encoding a polypeptide as set forth in SEQ ID No. 14-24;
(b) a polynucleotide having a sequence as set forth in any one of SEQ ID No. 1-13;
(c) a polynucleotide having a nucleotide sequence homology of 75% or more (preferably 85% or more, more preferably 90% or more or 95% or more or 98% or more or 99%) with any one of the sequences shown in SEQ ID No. 1-13;
(d) 1-13, or a polynucleotide in which 1-60 (preferably 1-30, more preferably 1-10) nucleotides are truncated or added at the 5 'end and/or 3' end of the polynucleotide shown in SEQ ID NO;
(e) a polynucleotide complementary to any one of the polynucleotides of (a) - (d).
In a preferred embodiment, the nucleic acid construct of the invention has a structure of formula III from 5 'to 3':
Z1-Z2-Z3 (III)
in the formula (I), the compound is shown in the specification,
Z1-Z3 are each an element used to construct the construct;
each "-" is independently a bond or a nucleotide linking sequence;
z1 is the coding sequence of signal peptide;
z2 is a linker sequence;
z3 is the coding sequence of the foreign protein;
wherein the coding sequence of the signal peptide is selected from the group consisting of:
(a) a polynucleotide encoding a polypeptide as set forth in SEQ ID No. 14-24;
(b) a polynucleotide having a sequence as set forth in any one of SEQ ID No. 1-13;
(c) a polynucleotide having a nucleotide sequence homology of 75% or more (preferably 85% or more, more preferably 90% or more or 95% or more or 98% or more or 99%) with any one of the sequences shown in SEQ ID No. 1-13;
(d) 1-13, or a polynucleotide in which 1-60 (preferably 1-30, more preferably 1-10) nucleotides are truncated or added at the 5 'end and/or 3' end of the polynucleotide shown in SEQ ID NO;
(e) a polynucleotide complementary to any one of the polynucleotides of (a) - (d).
In a preferred embodiment, the amino acid sequence of the signal peptide according to the invention has the sequence shown in SEQ ID NO. 14-24 or an active fragment thereof or has a homology of > 85%, preferably > 90% with the amino acid sequence shown in SEQ ID NO. 14-24; more preferably 95% homology or more; most preferably, homology of more than or equal to 97%, such as more than 98%, more than 99%) and has the same activity as the sequence shown in SEQ ID NO. 14-24.
In the present invention, the selection of the coding sequence of the foreign protein is not particularly limited, and generally, the coding sequence of the foreign protein encodes a protein selected from the group consisting of: luciferin protein, luciferase (e.g., firefly luciferase), green fluorescent protein, yellow fluorescent protein, aminoacyl tRNA synthetase, glyceraldehyde-3-phosphate dehydrogenase, catalase, actin, antibody or variable region thereof, luciferase mutant, or a combination thereof.
The coding sequence for the foreign protein may also encode a protein selected from the group consisting of: alpha-amylase, enteromycin A, hepatitis C virus E2 glycoprotein, insulin precursor, interferon alpha A, interleukin-1 beta, lysozyme, serum albumin, single chain antibody fragments (scFV), transthyretin, tyrosinase, xylanase, or a combination thereof.
In addition, the nucleic acid constructs of the invention may be linear or circular. The nucleic acid construct of the present invention may be single-stranded or double-stranded. The nucleic acid constructs of the invention may be DNA, RNA, or DNA/RNA hybrids.
Preferred signal peptide sequences of the invention and their nucleotide sequences encoding signal peptides are shown in Table 1.
In another preferred embodiment, said construct further comprises an element or a combination thereof selected from the group consisting of: promoters, terminators, poly (A) elements, transport elements, gene targeting elements, selectable marker genes, enhancers, resistance genes, transposase encoding genes.
A variety of selectable marker genes are applicable to the present invention, including but not limited to: auxotrophic markers, resistance markers, reporter gene markers. The use of a selectable marker serves to screen for recombinant cells (recombinants) so that recipient cells can be significantly distinguished from non-transformed cells. The auxotrophic marker is a marker gene that is introduced so as to complement a mutant gene of a recipient cell, thereby allowing the recipient cell to exhibit wild-type growth. The resistance marker refers to transferring resistance genes into receptor cells, and the transferred genes enable the receptor cells to show drug resistance at a certain drug concentration. As a preferred mode of the invention, a resistance marker is used to achieve convenient screening of recombinant cells.
In the invention, the nucleic acid construct is applied to an in vitro protein synthesis system, so that the translation efficiency of the foreign protein can be obviously improved. In a preferred embodiment, the use of the nucleic acid construct of the invention in the yeast in vitro protein synthesis system of the invention significantly increases the efficiency of protein translation.
Carrier
The invention also provides a vector or combination of vectors comprising the nucleic acid construct of the invention. Preferably, the carrier is selected from: bacterial plasmids, bacteriophages, yeast plasmids, or animal cell vectors, shuttle vectors. Further, the vector may be a transposon vector. Methods for preparing recombinant vectors are well known to those of ordinary skill in the art. Any plasmid and vector may be used as long as it can replicate and is stable in the host.
One of ordinary skill in the art can use well-known methods to construct expression vectors containing the promoter and/or gene sequences of interest described herein. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like.
Genetically engineered cell
The invention also provides a genetic engineering cell, wherein the genetic engineering cell contains the construct or the vector combination, or the chromosome of the genetic engineering cell is integrated with the construct or the vector. In another preferred embodiment, the genetically engineered cell further comprises a vector comprising a gene encoding a transposase or having a transposase gene integrated into its chromosome.
Preferably, the genetically engineered cell is a eukaryotic cell.
In another preferred embodiment, the eukaryotic cells, include, but are not limited to: a yeast cell (preferably, a kluyveromyces cell, more preferably a kluyveromyces lactis cell).
The constructs or vectors of the invention may be used to transform appropriate genetically engineered cells. The genetically engineered cells may be prokaryotic cells, such as E.coli, Streptomyces, Agrobacterium: or lower eukaryotic cells, such as yeast cells; or a higher animal cell, such as an insect cell. It is clear to one of ordinary skill in the art how to select appropriate vectors and genetically engineered cells. Transformation of genetically engineered cells with recombinant DNA may be carried out using conventional techniques well known to those skilled in the art. When the host is a prokaryote (e.g., Escherichia coli), CaCl may be used2The treatment can also be carried out by electroporation. When the host is a eukaryote, the following DNA may be usedThe transfection method comprises the following steps: calcium phosphate coprecipitation, conventional mechanical methods (e.g., microinjection, electroporation, liposome encapsulation, etc.). The transformed plant may be transformed by methods such as Agrobacterium transformation or biolistic transformation, for example, leaf disc method, immature embryo transformation, flower bud soaking method, etc.
In addition, the genetically engineered cells of the invention can be used to produce or provide the nucleic acid constructs of the invention.
In vitro high-flux protein synthesis method
The signal peptide and the construction containing the signal peptide coding sequence are particularly suitable for remarkably improving the synthesis efficiency or the yield of foreign proteins in an in vitro biosynthesis system.
Correspondingly, the invention also provides an in vitro high-flux protein synthesis method, which comprises the following steps:
(i) providing a nucleic acid construct according to the first aspect of the invention in the presence of an in vitro protein synthesis system;
(ii) (ii) incubating the in vitro protein synthesis system of step (i) under suitable conditions for a period of time T1, thereby synthesizing the foreign protein.
In another preferred example, the method further comprises: (iii) optionally isolating or detecting said foreign protein from said in vitro protein synthesis system.
The main advantages of the invention include:
(1) the invention discovers for the first time that the signal peptide related sequence and the coding sequence of the foreign protein are used as nucleic acid constructs, and the nucleic acid constructs can be applied to the in vitro protein synthesis system of the invention, and can be used for improving the translation efficiency of target protein and expressing and purifying the target protein.
(2) The signal peptide related sequence of the invention can affect the folding of mRNA after the translation initiation codon, thereby changing the translation efficiency of the target protein.
(3) Compared with other cells, the kluyveromyces lactis can be applied to the production of proteins in the fields of food and medicines due to the safety and high efficiency of the kluyveromyces lactis, and the in vitro protein synthesis system has the advantages of suitability for high-throughput protein synthesis screening, short toxic protein synthesis time, low cost and the like, so the in vitro protein synthesis system derived from the kluyveromyces lactis cells can be widely applied to related fields.
(4) The signal peptide related sequence provided by the invention can improve the translation efficiency of a target foreign protein, and can increase the possibility of synthesizing different proteins of a yeast in-vitro protein synthesis system (such as a Kluyveromyces lactis in-vitro protein synthesis system).
(5) The invention firstly develops a novel signal peptide for improving the protein translation efficiency of an in vitro protein synthesis system and a nucleic acid construct comprising a signal peptide coding sequence.
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are percentages and parts by weight.
Material
Unless otherwise specified, the materials and reagents used in the examples of the present invention are commercially available products.
The foreign proteins in the examples are exemplified by eGFP.
EXAMPLE 1 determination of eukaryotic Signal peptide related sequences
1.1 Source and determination of related sequences of Signal peptides: randomly intercepting a DNA sequence corresponding to the N end of the constructed foreign protein, and screening a sequence or an element which is obviously improved for the expression of the foreign protein through experiments.
Specifically, 30 nucleotide fragments with the length of 36,54 or other lengths are selected and synthesized, and partial bases are modified by adopting a mode of alternative substitution of synonymous codons, so as to improve the success rate of plasmid construction, wherein the method comprises the following steps (but is not limited to): to reduce the GC content of the signal peptide-related sequence and thereby reduce the annealing temperature of the sequence; or with preferred codons. Dozens of plasmids are obtained by construction, and the results of analysis and screening on 30 plasmids and testing the effect of the plasmids on the expression of foreign proteins show that compared with a control, 13 signal peptide related sequences have the effect of improving the expression of proteins through experimental verification, and the amino acid sequences of the corresponding signal peptides and the nucleotide sequence information of the coded signal peptides are listed in Table 1. Other sequence information that is not or not significantly effective in increasing protein expression is not listed.
TABLE 1 plasmids and related nucleic acid sequences
EXAMPLE 2 construction of plasmid for in vitro protein Synthesis System containing Signal peptide-related sequence
Construction of plasmid: for the selected 30 signal peptide related sequences and the linker sequences (containing TEV cleavage sites on the linker sequences) were amplified using 1 pair of primers, and the plasmid backbone originally containing the protein of interest (exemplified by eGFP) was amplified using its corresponding reverse primer. After amplification, 30 signal peptide related sequences + connecting sequence fragments are respectively inserted into the N end of the target protein. In the finally constructed plasmid, 30 signal peptide related sequence + linker nucleic acid sequences were inserted between the ATG start codon and eGFP of the pD2P-eGFP plasmid. The names of 13 plasmids are respectively: pD2P-1.0SP- (001-.
The specific construction process is as follows:
performing PCR amplification by using 2 pairs of primers respectively, and mixing 10 mu L of correctly identified amplification products; adding 0.5. mu.L of DpnI into 10. mu.L of the amplification product, and incubating at 37 ℃ for 6 h; adding 4 mu L of the product treated by the DpnI into 50 mu L of DH5 alpha competent cells, placing the cells on ice for 30min, thermally shocking the cells for 45s at 42 ℃, placing the cells on ice for 3min, adding 200 mu L of LB liquid culture medium, performing shake culture at 37 ℃ for 4h, and coating the cells on LB solid culture medium containing Amp antibiotics for overnight culture; 6 monoclonals are picked for amplification culture, sequencing is carried out to confirm correctness, and plasmids are extracted for storage.
EXAMPLE 3 use of Signal peptide-related sequences in vitro protein Synthesis systems
3.1 amplification of a fragment containing a signal peptide-related sequence between the transcription start and termination sequences of T7 and a fragment of pD2P-eGFP in all plasmids by PCR using the primers pD2P _ F: CGCGAAATTAATACGACTCACTATAGG (SEQ ID No.:27) and pD2P _ R: TCCGGATATAGTTCCTCCTTTCAG (SEQ ID No.: 28).
And purifying and enriching the amplified DNA fragment which is determined to be correct through sequencing by using an ethanol precipitation method: adding 1/10 volume of 3M sodium acetate (pH5.2) into the PCR product, adding 2.5-3 times volume of 95% ethanol, and incubating on ice for 15 min; centrifuging at room temperature at a speed higher than 14000g for 30min, and discarding the supernatant; washing with 70% ethanol, centrifuging for 15min, discarding the supernatant, dissolving the precipitate with ultrapure water, and determining the DNA concentration.
3.2 the purified DNA fragments were added to the home-made in vitro protein synthesis system according to the instructions. And placing the reaction system in an environment with the temperature of 22-30 ℃, and standing and incubating for about 2-6 h. Immediately after the reaction, the reaction mixture was placed in an Envision 2120 multifunctional microplate reader (Perkin Elmer), and read to detect the intensity of eGFP signal and Relative Fluorescence Unit (RFU) as an activity Unit.
PC (Positive control) is a group to which only a linker sequence was added to the N-terminus of the enhanced green fluorescent protein, and NC (negative control) is a group to which no nucleic acid construct was added. Mu.l, 2. mu.l and 3. mu.l are the amounts of the DNA template added to the in vitro protein synthesis system, respectively, and the total reaction system volume of all reactions is 30. mu.l.
Results of the experiment
1. Construction of plasmid for in vitro protein Synthesis System
Through a plurality of attempts, 30 in vitro protein synthesis system plasmids containing signal peptide related sequences are successfully constructed.
2. Application of signal peptide related sequence in vitro protein synthesis system
As shown in fig. 2, 13 related sequences including signal peptide all caused significant increase in RFU value of eGFP emission in vitro protein synthesis system (RFU value reached more than 1500 after 3 hours of reaction), which was as high as 2900. In particular, pD2P-1.0SP-012 (1. mu.l of DNA template was added, and the RFU value reached 2900 after 3 hours of reaction) showed a 2.6-fold increase in relative fluorescence unit value as compared with the control PC (RFU value of 800) to which no signal peptide-related sequence had been inserted.
For the other 17 unlisted signal peptide sequences, the relative fluorescence unit values were unchanged or not significantly changed from PC, mostly 800-830, e.g., 823 for pD2P-1.0SP-019 and 816 for pD2P-1.0 SP-027.
The result of the invention shows that the signal peptide related sequence at the N end of the target protein can obviously improve the yield of the target protein and greatly improve the expression and purification effects of the target protein. The translation efficiency of the target protein is improved, the selectivity of a protein expression and purification mode of an in vitro synthesis system is increased, and the availability of the in vitro protein synthesis system is greatly enhanced.
Further, the present inventors have found that the translation efficiency of a target protein can be further improved by combining 5'-UTR, a strong promoter (e.g., T7 promoter, T3 promoter, SP6 promoter), a different IRES element (e.g., KLNCE102) and a different signal peptide-related sequence, 3' -UTR, and the like.
COMPARATIVE EXAMPLE (PC and NC)
PC (Positive control) is an experimental group in which only a linker sequence was added to the N-terminus of the enhanced green fluorescent protein, and the RFU value of the foreign protein was 800 and the total volume of the reaction system was 30. mu.l when 1. mu.l of the DNA template was added.
NC (negative control) is an experimental group without any nucleic acid construct, the RFU value of the foreign protein is 20, and the total volume of the reaction system is 30. mu.l.
Wherein 1. mu.l, 2. mu.l and 3. mu.l in FIG. 2 represent the amounts of the DNA templates added to the in vitro protein synthesis system, respectively, the total reaction system volume of all reactions was 30. mu.l, and the reaction time was 3 hours.
All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.
Reference documents:
1.Fromm HJ,Hargrove M.Essentials of Biochemistry.2012.
2.Garcia RA,Riley MR.Applied biochemistry and biotechnology.Humana Press,;1981.263-264p.
3.Martoglio B.Intramembrane proteolysis and post-targeting functions of signal peptides.Biochem Soc Trans.2003;31(6):1243–7.
4.Owji H,Nezafat N,Negahdaripour M,Hajiebrahimi A,Ghasemi Y.A Comprehensive Review of Signal Peptides:Structure,Roles,and Applications.Eur J Cell Biol.2018.
5.Liu H,Wu R,Yuan L,Tian G,Huang X,Wen Y,et al.Introducing a cleavable signal peptide enhances the packaging efficiency of lentiviral vectors pseudotyped with Japanese encephalitis virus envelope proteins.Virus Res.2017;229:9–16.
6.Cui Y,Meng Y,Zhang J,Cheng B,Yin H,Gao C,et al.Efficient secretory expression of recombinant proteins in Escherichia coli with a novel actinomycete signal peptide.Protein Expr Purif.2017;129:69–74.
7.Ling HL,Rahmat Z,Murad AMA,Mahadi NM,Illias RM.Proteome-based identification of signal peptides for improved secretion of recombinant cyclomaltodextrin glucanotransferase in Escherichia coli.Process Biochem.2017;61:47–55.
8.Zhang S,Corin K.18-Peptide surfactants in membrane protein purification and stabilization A2-Koutsopoulos,Sotirios BT-Peptide Applications in Biomedicine,Biotechnology and Bioengineering.In Woodhead Publishing;2018.p.485–512.
9.Stone TA,Deber CM.Therapeutic design of peptide modulators of protein-protein interactions in membranes.Biochim Biophys Acta-Biomembr.2017;1859(4):577–85.
10.Katzen F,Chang G,Kudlicki W.The past,present and future of cell-free protein synthesis.Trends Biotechnol.2005;23(3):150–6.
11.Gan R,Jewett MC.A combined cell-free transcription-translation system from Saccharomyces cerevisiae for rapid and robust protein synthesis.Biotechnol J.2014;9(5):641–51.
12.Lu Y.Cell-free synthetic biology:Engineering in an open world.Synth Syst Biotechnol.2017;2(1):23–7.
13.Kralicek A V.,Radjainia M,Mohamad Ali NAB,Carraher C,Newcomb RD,Mitra AK.A PCR-directed cell-free approach to optimize protein expression using diverse fusion tags.Protein Expr Purif.2011;80(1):117–24.
14.Hansted JG,L,F,Sperl ing-Petersen HU,Mortensen KK.Expressivity tag:A novel tool for increased expression in Escherichia coli.J Biotechnol.2011;155(3):275–83.
15.Kasi D,Nah HJ,Catherine C,Kim ES,Han K,Ha JC,et al.Enhanced Production of Soluble Recombinant Proteins With an In Situ-Removable Fusion Partner in a Cell-Free Synthesis System.Biotechnol J.2017;12(11):1–6.
16.S,Nordlund P,Weigelt J,Hal lberg BM,Bray J,Gileadi O,et al.Protein production and purification.Nat Methods.2008;5(2):135–46.
sequence listing
<110> Kangma (Shanghai) Biotech Co., Ltd
<120> signal peptide related sequence and application thereof in protein synthesis
<130> P2018-1332
<141> 2018-08-07
<160> 28
<170> SIPOSequenceListing 1.0
<210> 1
<211> 36
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 1
agtgagcaaa gccaattaga tgattcgact atagac 36
<210> 2
<211> 36
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 2
ctgacaactg ttctccctaa cgtagctaca ttaaac 36
<210> 3
<211> 54
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 3
atgctgacaa ctgttctccc taacgtagct acattaaaca gtatgtttgc cctg 54
<210> 4
<211> 36
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 4
aattgctccg cacattgtat caaaaaggct ttacct 36
<210> 5
<211> 54
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 5
aattgctccg cacattgtat caaaaaggct ttacctgcac agtggatccg ttgc 54
<210> 6
<211> 36
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 6
aaaacacata tagtcagctc agtaacaaca acacta 36
<210> 7
<211> 54
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 7
aaaacacata tagtcagctc agtaacaaca acactattgc taggttccat atta 54
<210> 8
<211> 36
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 8
tctggtggtc aaattttcgt aaagacgctg accggt 36
<210> 9
<211> 36
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 9
tctggtggtc aaattttcgt caaaactcta acaggt 36
<210> 10
<211> 36
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 10
tctggtggtc aaattttcgt taaaactctt actggt 36
<210> 11
<211> 27
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 11
aagcctccag tatacccatc gatttgc 27
<210> 12
<211> 36
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 12
atgattacag aaacatcatc accgttcaga tctata 36
<210> 13
<211> 54
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 13
atggtcgcta gaggtagaac agacgagata tctacagatg tttcagaggc taat 54
<210> 14
<211> 12
<212> PRT
<213> Artificial sequence (artificial sequence)
<400> 14
Ser Glu Gln Ser Gln Leu Asp Asp Ser Thr Ile Asp
1 5 10
<210> 15
<211> 12
<212> PRT
<213> Artificial sequence (artificial sequence)
<400> 15
Leu Thr Thr Val Leu Pro Asn Val Ala Thr Leu Asn
1 5 10
<210> 16
<211> 17
<212> PRT
<213> Artificial sequence (artificial sequence)
<400> 16
Leu Thr Thr Val Leu Pro Asn Val Ala Thr Leu Asn Ser Met Phe Ala
1 5 10 15
Leu
<210> 17
<211> 12
<212> PRT
<213> Artificial sequence (artificial sequence)
<400> 17
Asn Cys Ser Ala His Cys Ile Lys Lys Ala Leu Pro
1 5 10
<210> 18
<211> 18
<212> PRT
<213> Artificial sequence (artificial sequence)
<400> 18
Asn Cys Ser Ala His Cys Ile Lys Lys Ala Leu Pro Ala Gln Trp Ile
1 5 10 15
Arg Cys
<210> 19
<211> 12
<212> PRT
<213> Artificial sequence (artificial sequence)
<400> 19
Lys Thr His Ile Val Ser Ser Val Thr Thr Thr Leu
1 5 10
<210> 20
<211> 18
<212> PRT
<213> Artificial sequence (artificial sequence)
<400> 20
Lys Thr His Ile Val Ser Ser Val Thr Thr Thr Leu Leu Leu Gly Ser
1 5 10 15
Ile Leu
<210> 21
<211> 12
<212> PRT
<213> Artificial sequence (artificial sequence)
<400> 21
Ser Gly Gly Gln Ile Phe Val Lys Thr Leu Thr Gly
1 5 10
<210> 22
<211> 9
<212> PRT
<213> Artificial sequence (artificial sequence)
<400> 22
Lys Pro Pro Val Tyr Pro Ser Ile Cys
1 5
<210> 23
<211> 11
<212> PRT
<213> Artificial sequence (artificial sequence)
<400> 23
Ile Thr Glu Thr Ser Ser Pro Phe Arg Ser Ile
1 5 10
<210> 24
<211> 17
<212> PRT
<213> Artificial sequence (artificial sequence)
<400> 24
Val Ala Arg Gly Arg Thr Asp Glu Ile Ser Thr Asp Val Ser Glu Ala
1 5 10 15
Asn
<210> 25
<211> 48
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 25
gaaaacctgt atttccaagg aggtagtgga ggaagtggtg gaagtgga 48
<210> 26
<211> 16
<212> PRT
<213> Artificial sequence (artificial sequence)
<400> 26
Glu Asn Leu Tyr Phe Gln Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly
1 5 10 15
<210> 27
<211> 27
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 27
cgcgaaatta atacgactca ctatagg 27
<210> 28
<211> 24
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 28
tccggatata gttcctcctt tcag 24
Claims (14)
1. A nucleic acid construct comprising a first nucleotide sequence encoding a signal peptide operably linked to a second nucleotide sequence encoding a foreign protein, the 3' end of the first nucleotide sequence being upstream of the second nucleotide sequence, and the first nucleotide sequence being a nucleotide sequence encoding a signal peptide: the amino acid sequence is ITETSSPFRSI signal peptide.
2. The nucleic acid construct of claim 1, wherein: the operable linkage is direct linkage or linkage through a linking sequence.
3. The nucleic acid construct of claim 2, wherein: the connecting sequence is SEQ ID No.: 25.
4. A signal peptide, characterized by: the amino acid sequence of the polypeptide is encoded by the first nucleotide sequence of claim 1.
5. A vector or vector combination comprising the nucleic acid construct of any one of claims 1 to 3.
6. A genetically engineered cell having the nucleic acid construct of any one of claims 1 to 3 integrated at one or more sites in the genome of the genetically engineered cell, or having the vector or combination of vectors of claim 5 incorporated therein;
the genetically engineered cell is selected from a prokaryotic cell, a Chinese hamster ovary cell, an insect cell, a rabbit reticulocyte, a yeast cell, or a combination thereof.
7. A kit comprising reagents selected from one or more of the group consisting of:
(a) the nucleic acid construct of any one of claims 1-3;
(b) the vector or combination of vectors of claim 5; and
(c) the genetically engineered cell of claim 6.
8. The kit of claim 7, further comprising (d) an in vitro biosynthetic system.
9. The kit of claim 8, wherein: the in vitro biosynthetic system is selected from the group consisting of: a yeast in vitro biosynthesis system, a chinese hamster ovary cell in vitro biosynthesis system, an insect cell in vitro biosynthesis system, a Hela cell in vitro biosynthesis system, or a combination thereof.
10. The kit of claim 9, wherein: the yeast in-vitro biosynthesis system is a Kluyveromyces in-vitro biosynthesis system.
11. Use of a nucleic acid construct according to any of claims 1 to 3, a signal peptide according to claim 4, a vector or combination of vectors according to claim 5, a genetically engineered cell according to claim 6 or a kit according to any of claims 7 to 9 in an in vitro protein synthesis system.
12. An in vitro protein synthesis method, comprising the steps of:
(i) providing an in vitro biosynthetic system comprising the nucleic acid construct of any of claims 1-3;
(ii) (ii) incubating the in vitro biosynthetic system of step (i) under suitable conditions for a reaction time, thereby synthesizing the foreign protein.
13. The in vitro protein synthesis method according to claim 12, wherein the suitable conditions are a reaction temperature of 20-37 ℃ and a reaction time of 1-72 h.
14. The in vitro protein synthesis method of claim 12, further comprising: (iii) isolating or detecting the foreign protein.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202110864773.4A CN113528574B (en) | 2018-08-07 | 2018-08-07 | Signal peptide related sequence and application thereof in protein synthesis |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202110864773.4A CN113528574B (en) | 2018-08-07 | 2018-08-07 | Signal peptide related sequence and application thereof in protein synthesis |
CN201810888184.8A CN110819647A (en) | 2018-08-07 | 2018-08-07 | Signal peptide related sequence and application thereof in protein synthesis |
Related Parent Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201810888184.8A Division CN110819647A (en) | 2018-08-07 | 2018-08-07 | Signal peptide related sequence and application thereof in protein synthesis |
Publications (2)
Publication Number | Publication Date |
---|---|
CN113528574A true CN113528574A (en) | 2021-10-22 |
CN113528574B CN113528574B (en) | 2022-06-21 |
Family
ID=69533681
Family Applications (8)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202110866625.6A Active CN113481226B (en) | 2018-08-07 | 2018-08-07 | Signal peptide related sequence and application thereof in protein synthesis |
CN202110866617.1A Active CN113584059B (en) | 2018-08-07 | 2018-08-07 | Signal peptide related sequence and application thereof in protein synthesis |
CN202110866626.0A Active CN113528575B (en) | 2018-08-07 | 2018-08-07 | Signal peptide related sequence and application thereof in protein synthesis |
CN202110866634.5A Active CN113584060B (en) | 2018-08-07 | 2018-08-07 | Signal peptide related sequence and application thereof in protein synthesis |
CN202110866616.7A Active CN113584058B (en) | 2018-08-07 | 2018-08-07 | Signal peptide related sequence and application thereof in protein synthesis |
CN201810888184.8A Pending CN110819647A (en) | 2018-08-07 | 2018-08-07 | Signal peptide related sequence and application thereof in protein synthesis |
CN202110876455.XA Active CN113667685B (en) | 2018-08-07 | 2018-08-07 | Signal peptide related sequence and application thereof in protein synthesis |
CN202110864773.4A Active CN113528574B (en) | 2018-08-07 | 2018-08-07 | Signal peptide related sequence and application thereof in protein synthesis |
Family Applications Before (7)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202110866625.6A Active CN113481226B (en) | 2018-08-07 | 2018-08-07 | Signal peptide related sequence and application thereof in protein synthesis |
CN202110866617.1A Active CN113584059B (en) | 2018-08-07 | 2018-08-07 | Signal peptide related sequence and application thereof in protein synthesis |
CN202110866626.0A Active CN113528575B (en) | 2018-08-07 | 2018-08-07 | Signal peptide related sequence and application thereof in protein synthesis |
CN202110866634.5A Active CN113584060B (en) | 2018-08-07 | 2018-08-07 | Signal peptide related sequence and application thereof in protein synthesis |
CN202110866616.7A Active CN113584058B (en) | 2018-08-07 | 2018-08-07 | Signal peptide related sequence and application thereof in protein synthesis |
CN201810888184.8A Pending CN110819647A (en) | 2018-08-07 | 2018-08-07 | Signal peptide related sequence and application thereof in protein synthesis |
CN202110876455.XA Active CN113667685B (en) | 2018-08-07 | 2018-08-07 | Signal peptide related sequence and application thereof in protein synthesis |
Country Status (1)
Country | Link |
---|---|
CN (8) | CN113481226B (en) |
Families Citing this family (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN111484998B (en) | 2019-05-30 | 2023-04-21 | 康码(上海)生物科技有限公司 | Method for in vitro quantitative co-expression of multiple proteins and application thereof |
US20230134868A1 (en) | 2019-11-30 | 2023-05-04 | Kangma-Healthcode (Shanghai) Biotech Co., Ltd | Biomagnetic microsphere and preparation method therefor and use thereof |
CN113402589A (en) * | 2021-06-18 | 2021-09-17 | 苏州工业园区唯可达生物科技有限公司 | Signal peptide for improving antibody yield |
CN116370335A (en) | 2021-12-31 | 2023-07-04 | 康码(上海)生物科技有限公司 | Virus protection solution for wiping and virus protection wiping product |
CN117683804A (en) * | 2022-09-09 | 2024-03-12 | 康码(上海)生物科技有限公司 | Nucleic acid construct and application thereof in IVTT system |
Citations (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101376888A (en) * | 2008-09-28 | 2009-03-04 | 李校堃 | Method for producing secretion expression recombinant human fibroblast growth factor-21 |
CN101386868A (en) * | 2008-09-23 | 2009-03-18 | 复旦大学 | Method for improving expression level of recombinant protein in kluyveromyces |
WO2010085012A1 (en) * | 2009-01-23 | 2010-07-29 | 한국과학기술연구원 | Method for secreting and producing foreign protein in e. coli |
CN103173367A (en) * | 2013-03-11 | 2013-06-26 | 江南大学 | Engineered Saccharomyces cerevisiae producing heat-stability recombinant trypsin, and its application |
CN104059128A (en) * | 2010-09-17 | 2014-09-24 | 上海凯茂生物医药有限公司 | Artificially synthesized signal sequence and application thereof |
JP2014223064A (en) * | 2013-04-24 | 2014-12-04 | 東ソー株式会社 | Signal peptide and method for producing proteins using the same |
CN107474142A (en) * | 2017-09-04 | 2017-12-15 | 中国动物疫病预防控制中心 | Promote polypeptide and its relevant biological material and the application of destination protein secretion |
WO2018081350A1 (en) * | 2016-10-27 | 2018-05-03 | Medimmune, Llc | Signal polypeptide for improved secretion of protein |
Family Cites Families (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US7863020B2 (en) * | 2000-06-28 | 2011-01-04 | Glycofi, Inc. | Production of sialylated N-glycans in lower eukaryotes |
JP5028551B2 (en) * | 2003-11-21 | 2012-09-19 | フェネックス インコーポレイテッド | Expression system improved by SEC secretion |
CN101250551B (en) * | 2008-04-08 | 2011-09-14 | 上海师范大学 | Expression vector for improving soy protein content and quality as well as preparation and use thereof |
CN102993292A (en) * | 2012-12-14 | 2013-03-27 | 上海柯莱逊生物技术有限公司 | AFP (Alpha Fetal Protein) recombinant protein and in-intro recombinant expression method |
DK3475295T3 (en) * | 2016-06-24 | 2022-10-24 | Scripps Research Inst | Novel nucleoside triphosphate transporter and uses thereof |
MX2018015596A (en) * | 2016-10-11 | 2019-03-14 | Wellstat Ophthalmics Corp | Fusion protein between short form rod-derived cone viability factor and a hydrophilic peptide. |
CN107936096A (en) * | 2017-10-23 | 2018-04-20 | 华南理工大学 | A kind of signal peptide that can effectively improve protein secretion efficiency and its application |
-
2018
- 2018-08-07 CN CN202110866625.6A patent/CN113481226B/en active Active
- 2018-08-07 CN CN202110866617.1A patent/CN113584059B/en active Active
- 2018-08-07 CN CN202110866626.0A patent/CN113528575B/en active Active
- 2018-08-07 CN CN202110866634.5A patent/CN113584060B/en active Active
- 2018-08-07 CN CN202110866616.7A patent/CN113584058B/en active Active
- 2018-08-07 CN CN201810888184.8A patent/CN110819647A/en active Pending
- 2018-08-07 CN CN202110876455.XA patent/CN113667685B/en active Active
- 2018-08-07 CN CN202110864773.4A patent/CN113528574B/en active Active
Patent Citations (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101386868A (en) * | 2008-09-23 | 2009-03-18 | 复旦大学 | Method for improving expression level of recombinant protein in kluyveromyces |
CN101376888A (en) * | 2008-09-28 | 2009-03-04 | 李校堃 | Method for producing secretion expression recombinant human fibroblast growth factor-21 |
WO2010085012A1 (en) * | 2009-01-23 | 2010-07-29 | 한국과학기술연구원 | Method for secreting and producing foreign protein in e. coli |
CN104059128A (en) * | 2010-09-17 | 2014-09-24 | 上海凯茂生物医药有限公司 | Artificially synthesized signal sequence and application thereof |
CN103173367A (en) * | 2013-03-11 | 2013-06-26 | 江南大学 | Engineered Saccharomyces cerevisiae producing heat-stability recombinant trypsin, and its application |
JP2014223064A (en) * | 2013-04-24 | 2014-12-04 | 東ソー株式会社 | Signal peptide and method for producing proteins using the same |
WO2018081350A1 (en) * | 2016-10-27 | 2018-05-03 | Medimmune, Llc | Signal polypeptide for improved secretion of protein |
CN107474142A (en) * | 2017-09-04 | 2017-12-15 | 中国动物疫病预防控制中心 | Promote polypeptide and its relevant biological material and the application of destination protein secretion |
Non-Patent Citations (2)
Title |
---|
LARS KOBER ET AL: "Optimized Signal Peptides for the Development of High Expressing CHO Cell Lines", 《BIOTECHNOLOGY AND BIOENGINEERING》 * |
王庆华等: "影响毕赤酵母高效表达重组蛋白的主要因素及其研究进展", 《药学学报》 * |
Also Published As
Publication number | Publication date |
---|---|
CN110819647A (en) | 2020-02-21 |
CN113528575A (en) | 2021-10-22 |
CN113584060B (en) | 2023-02-07 |
CN113528574B (en) | 2022-06-21 |
CN113667685A (en) | 2021-11-19 |
CN113584058A (en) | 2021-11-02 |
CN113667685B (en) | 2023-02-28 |
CN113584058B (en) | 2023-02-10 |
CN113584059A (en) | 2021-11-02 |
CN113528575B (en) | 2022-06-21 |
CN113481226A (en) | 2021-10-08 |
CN113481226B (en) | 2022-06-21 |
CN113584059B (en) | 2023-02-10 |
CN113584060A (en) | 2021-11-02 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN113528574B (en) | Signal peptide related sequence and application thereof in protein synthesis | |
CN110408635B (en) | Application of nucleic acid construct containing streptavidin element in protein expression and purification | |
CN110408636B (en) | DNA sequence with multiple labels connected in series and application thereof in protein expression and purification system | |
JP7292442B2 (en) | Modified aminoacyl-tRNA synthetase and uses thereof | |
CN111778169B (en) | Method for improving in vitro protein synthesis efficiency | |
AU2020242724B2 (en) | Aminoacyl-tRNA synthetase for efficiently introducing lysine derivative in protein | |
CN110551745A (en) | Multiple histidine sequence tag and application thereof in protein expression and purification | |
CN114555812A (en) | Materials and methods for protein production | |
CN111850020B (en) | Introduction of unnatural amino acids in proteins using plasmid systems | |
CN110938649A (en) | Protein synthesis system for improving expression quantity of foreign protein and application method thereof | |
CN111378047B (en) | Fusion tag protein for improving protein expression and application thereof | |
JP2018514231A (en) | Separation of growth and protein production | |
JP7028986B2 (en) | Tandem DNA element that can increase protein synthesis efficiency | |
KR101373297B1 (en) | Expression Vector Comprising Gene coding for E. coli Phosphoglycerate kinase As a Novel Fusion Partner | |
CN114057861B (en) | bio-PROTAC artificial protein targeting UBE2C | |
JP2023509578A (en) | Polypeptide tags and their use in in vitro protein synthesis | |
US20090035815A1 (en) | Synthetic Gene for Enhanced Expression in E. Coli | |
WO2024051855A1 (en) | Nucleic acid construct and use thereof in ivtt system | |
RU2803949C1 (en) | Method for crm197 protein expression | |
CA3233224A1 (en) | Chimeric protein and expression system | |
CN116987683A (en) | O-methyltransferase for catalyzing Parsley phenol to generate osthole, and encoding gene and application thereof | |
KR100755727B1 (en) | - Fragment Peptide of -Galactosidase as a Fusion Partner and Preparation Method of Recombinant Protein Using the Same |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
GR01 | Patent grant | ||
GR01 | Patent grant | ||
PE01 | Entry into force of the registration of the contract for pledge of patent right | ||
PE01 | Entry into force of the registration of the contract for pledge of patent right |
Denomination of invention: Signal peptide related sequences and their applications in protein synthesis Granted publication date: 20220621 Pledgee: China Construction Bank Corporation Shanghai Fengxian Branch Pledgor: KANGMA-HEATHCODE (SHANGHAI) BIOTECH Co.,Ltd. Registration number: Y2024980016719 |