TW202128735A - 源自cho細胞之蛋白分泌因子和包含彼之表現載體 - Google Patents
源自cho細胞之蛋白分泌因子和包含彼之表現載體 Download PDFInfo
- Publication number
- TW202128735A TW202128735A TW109142396A TW109142396A TW202128735A TW 202128735 A TW202128735 A TW 202128735A TW 109142396 A TW109142396 A TW 109142396A TW 109142396 A TW109142396 A TW 109142396A TW 202128735 A TW202128735 A TW 202128735A
- Authority
- TW
- Taiwan
- Prior art keywords
- seq
- protein
- acid sequence
- factor
- target protein
- Prior art date
Links
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 233
- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 203
- 239000013604 expression vector Substances 0.000 title claims abstract description 43
- 230000003248 secreting effect Effects 0.000 title abstract description 8
- 210000004027 cell Anatomy 0.000 claims abstract description 69
- 150000007523 nucleic acids Chemical group 0.000 claims abstract description 48
- 108091028043 Nucleic acid sequence Proteins 0.000 claims abstract description 42
- 230000014509 gene expression Effects 0.000 claims abstract description 42
- 210000004978 chinese hamster ovary cell Anatomy 0.000 claims abstract description 24
- 238000004519 manufacturing process Methods 0.000 claims abstract description 7
- 230000028327 secretion Effects 0.000 claims description 111
- 150000001413 amino acids Chemical group 0.000 claims description 63
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 claims description 21
- 238000003776 cleavage reaction Methods 0.000 claims description 21
- 238000000034 method Methods 0.000 claims description 21
- 230000007017 scission Effects 0.000 claims description 21
- 239000001963 growth medium Substances 0.000 claims description 15
- 102000004414 Calcitonin Gene-Related Peptide Human genes 0.000 claims description 6
- 108090000932 Calcitonin Gene-Related Peptide Proteins 0.000 claims description 6
- 108010017213 Granulocyte-Macrophage Colony-Stimulating Factor Proteins 0.000 claims description 6
- 102100039620 Granulocyte-macrophage colony-stimulating factor Human genes 0.000 claims description 6
- 102000010780 Platelet-Derived Growth Factor Human genes 0.000 claims description 6
- 108010038512 Platelet-Derived Growth Factor Proteins 0.000 claims description 6
- 108060008682 Tumor Necrosis Factor Proteins 0.000 claims description 6
- -1 cell Factors Proteins 0.000 claims description 6
- 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 claims description 6
- 102000003390 tumor necrosis factor Human genes 0.000 claims description 6
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 claims description 5
- 108020001507 fusion proteins Proteins 0.000 claims description 5
- 102000037865 fusion proteins Human genes 0.000 claims description 5
- 239000004475 Arginine Substances 0.000 claims description 4
- 102000053187 Glucuronidase Human genes 0.000 claims description 4
- 108010060309 Glucuronidase Proteins 0.000 claims description 4
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 claims description 4
- 239000012228 culture supernatant Substances 0.000 claims description 4
- ZIIUUSVHCHPIQD-UHFFFAOYSA-N 2,4,6-trimethyl-N-[3-(trifluoromethyl)phenyl]benzenesulfonamide Chemical compound CC1=CC(C)=CC(C)=C1S(=O)(=O)NC1=CC=CC(C(F)(F)F)=C1 ZIIUUSVHCHPIQD-UHFFFAOYSA-N 0.000 claims description 3
- 102000055025 Adenosine deaminases Human genes 0.000 claims description 3
- 108700040115 Adenosine deaminases Proteins 0.000 claims description 3
- 102100033367 Appetite-regulating hormone Human genes 0.000 claims description 3
- 102000007347 Apyrase Human genes 0.000 claims description 3
- 108010007730 Apyrase Proteins 0.000 claims description 3
- 108700016171 Aspartate ammonia-lyases Proteins 0.000 claims description 3
- 108010015428 Bilirubin oxidase Proteins 0.000 claims description 3
- 102000004506 Blood Proteins Human genes 0.000 claims description 3
- 108010017384 Blood Proteins Proteins 0.000 claims description 3
- 102000055006 Calcitonin Human genes 0.000 claims description 3
- 108060001064 Calcitonin Proteins 0.000 claims description 3
- 108010053835 Catalase Proteins 0.000 claims description 3
- 108090000317 Chymotrypsin Proteins 0.000 claims description 3
- 108010092674 Enkephalins Proteins 0.000 claims description 3
- 102000003951 Erythropoietin Human genes 0.000 claims description 3
- 108090000394 Erythropoietin Proteins 0.000 claims description 3
- 102000002464 Galactosidases Human genes 0.000 claims description 3
- 108010093031 Galactosidases Proteins 0.000 claims description 3
- 108010015776 Glucose oxidase Proteins 0.000 claims description 3
- 239000004366 Glucose oxidase Substances 0.000 claims description 3
- 102000004366 Glucosidases Human genes 0.000 claims description 3
- 108010056771 Glucosidases Proteins 0.000 claims description 3
- 102000004547 Glucosylceramidase Human genes 0.000 claims description 3
- 108010017544 Glucosylceramidase Proteins 0.000 claims description 3
- 102000002265 Human Growth Hormone Human genes 0.000 claims description 3
- 108010000521 Human Growth Hormone Proteins 0.000 claims description 3
- 239000000854 Human Growth Hormone Substances 0.000 claims description 3
- 108060003951 Immunoglobulin Proteins 0.000 claims description 3
- 102000008394 Immunoglobulin Fragments Human genes 0.000 claims description 3
- 108010021625 Immunoglobulin Fragments Proteins 0.000 claims description 3
- 102000004877 Insulin Human genes 0.000 claims description 3
- 108090001061 Insulin Proteins 0.000 claims description 3
- 102000006992 Interferon-alpha Human genes 0.000 claims description 3
- 108010047761 Interferon-alpha Proteins 0.000 claims description 3
- 102000003996 Interferon-beta Human genes 0.000 claims description 3
- 108090000467 Interferon-beta Proteins 0.000 claims description 3
- 102000008070 Interferon-gamma Human genes 0.000 claims description 3
- 108010074328 Interferon-gamma Proteins 0.000 claims description 3
- URLZCHNOLZSCCA-VABKMULXSA-N Leu-enkephalin Chemical compound C([C@@H](C(=O)N[C@@H](CC(C)C)C(O)=O)NC(=O)CNC(=O)CNC(=O)[C@@H](N)CC=1C=CC(O)=CC=1)C1=CC=CC=C1 URLZCHNOLZSCCA-VABKMULXSA-N 0.000 claims description 3
- 102000004882 Lipase Human genes 0.000 claims description 3
- 108090001060 Lipase Proteins 0.000 claims description 3
- 239000004367 Lipase Substances 0.000 claims description 3
- 108010064785 Phospholipases Proteins 0.000 claims description 3
- 102000015439 Phospholipases Human genes 0.000 claims description 3
- 108010057464 Prolactin Proteins 0.000 claims description 3
- 102000003946 Prolactin Human genes 0.000 claims description 3
- 102000019197 Superoxide Dismutase Human genes 0.000 claims description 3
- 108010012715 Superoxide dismutase Proteins 0.000 claims description 3
- 102000003425 Tyrosinase Human genes 0.000 claims description 3
- 108060008724 Tyrosinase Proteins 0.000 claims description 3
- 108010092464 Urate Oxidase Proteins 0.000 claims description 3
- BBBFJLBPOGFECG-VJVYQDLKSA-N calcitonin Chemical compound N([C@H](C(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H]([C@@H](C)O)C(=O)N1[C@@H](CCC1)C(N)=O)C(C)C)C(=O)[C@@H]1CSSC[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)O)C(=O)N1 BBBFJLBPOGFECG-VJVYQDLKSA-N 0.000 claims description 3
- 229960004015 calcitonin Drugs 0.000 claims description 3
- 230000021164 cell adhesion Effects 0.000 claims description 3
- 229960002376 chymotrypsin Drugs 0.000 claims description 3
- 210000004748 cultured cell Anatomy 0.000 claims description 3
- 230000004069 differentiation Effects 0.000 claims description 3
- 229940105423 erythropoietin Drugs 0.000 claims description 3
- 108010077689 gamma-aminobutyryl-2-methyltryptophyl-2-methyltryptophyl-2-methyltryptophyl-lysinamide Proteins 0.000 claims description 3
- 229940044627 gamma-interferon Drugs 0.000 claims description 3
- 229940116332 glucose oxidase Drugs 0.000 claims description 3
- 235000019420 glucose oxidase Nutrition 0.000 claims description 3
- 239000003102 growth factor Substances 0.000 claims description 3
- 229940088597 hormone Drugs 0.000 claims description 3
- 239000005556 hormone Substances 0.000 claims description 3
- 102000018358 immunoglobulin Human genes 0.000 claims description 3
- 229940125396 insulin Drugs 0.000 claims description 3
- 235000019421 lipase Nutrition 0.000 claims description 3
- OXCMYAYHXIHQOA-UHFFFAOYSA-N potassium;[2-butyl-5-chloro-3-[[4-[2-(1,2,4-triaza-3-azanidacyclopenta-1,4-dien-5-yl)phenyl]phenyl]methyl]imidazol-4-yl]methanol Chemical compound [K+].CCCCC1=NC(Cl)=C(CO)N1CC1=CC=C(C=2C(=CC=CC=2)C2=N[N-]N=N2)C=C1 OXCMYAYHXIHQOA-UHFFFAOYSA-N 0.000 claims description 3
- 229940097325 prolactin Drugs 0.000 claims description 3
- 230000002992 thymic effect Effects 0.000 claims description 3
- 102000011022 Chorionic Gonadotropin Human genes 0.000 claims description 2
- 108010062540 Chorionic Gonadotropin Proteins 0.000 claims description 2
- 108090000723 Insulin-Like Growth Factor I Proteins 0.000 claims description 2
- 102000005726 Pituitary Hormone-Releasing Hormones Human genes 0.000 claims description 2
- 108010031037 Pituitary Hormone-Releasing Hormones Proteins 0.000 claims description 2
- 102000013275 Somatomedins Human genes 0.000 claims description 2
- 229940015047 chorionic gonadotropin Drugs 0.000 claims description 2
- 239000002303 hypothalamus releasing factor Substances 0.000 claims description 2
- 102000016938 Catalase Human genes 0.000 claims 2
- 102000003978 Tissue Plasminogen Activator Human genes 0.000 claims 2
- 108090000373 Tissue Plasminogen Activator Proteins 0.000 claims 2
- 229960000187 tissue plasminogen activator Drugs 0.000 claims 2
- 102000004452 Arginase Human genes 0.000 claims 1
- 108700024123 Arginases Proteins 0.000 claims 1
- 102000004190 Enzymes Human genes 0.000 claims 1
- 108090000790 Enzymes Proteins 0.000 claims 1
- 108010086677 Gonadotropins Proteins 0.000 claims 1
- 102000006771 Gonadotropins Human genes 0.000 claims 1
- 102000005755 Intercellular Signaling Peptides and Proteins Human genes 0.000 claims 1
- 108010070716 Intercellular Signaling Peptides and Proteins Proteins 0.000 claims 1
- 229940088598 enzyme Drugs 0.000 claims 1
- 239000002622 gonadotropin Substances 0.000 claims 1
- 239000003630 growth substance Substances 0.000 claims 1
- 230000002267 hypothalamic effect Effects 0.000 claims 1
- 235000018102 proteins Nutrition 0.000 description 104
- 108010076504 Protein Sorting Signals Proteins 0.000 description 59
- 102000040430 polynucleotide Human genes 0.000 description 34
- 108091033319 polynucleotide Proteins 0.000 description 34
- 239000002157 polynucleotide Substances 0.000 description 34
- 239000013598 vector Substances 0.000 description 26
- 108090000765 processed proteins & peptides Proteins 0.000 description 23
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 14
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 14
- 102000004196 processed proteins & peptides Human genes 0.000 description 12
- 239000002609 medium Substances 0.000 description 11
- 229920001184 polypeptide Polymers 0.000 description 11
- 210000004899 c-terminal region Anatomy 0.000 description 8
- 230000010354 integration Effects 0.000 description 8
- 238000005516 engineering process Methods 0.000 description 7
- 108020004414 DNA Proteins 0.000 description 5
- 235000001014 amino acid Nutrition 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- 230000008569 process Effects 0.000 description 5
- 239000000126 substance Substances 0.000 description 5
- 230000009466 transformation Effects 0.000 description 5
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- 239000012634 fragment Substances 0.000 description 4
- 230000002068 genetic effect Effects 0.000 description 4
- 102000039446 nucleic acids Human genes 0.000 description 4
- 108020004707 nucleic acids Proteins 0.000 description 4
- 229960002621 pembrolizumab Drugs 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 238000013518 transcription Methods 0.000 description 4
- 230000035897 transcription Effects 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- 102000004225 Cathepsin B Human genes 0.000 description 3
- 108090000712 Cathepsin B Proteins 0.000 description 3
- 102000019034 Chemokines Human genes 0.000 description 3
- 108010012236 Chemokines Proteins 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- 101150003028 Hprt1 gene Proteins 0.000 description 3
- 108010013563 Lipoprotein Lipase Proteins 0.000 description 3
- 102000043296 Lipoprotein lipases Human genes 0.000 description 3
- 102000018098 Nucleobindin-2 Human genes 0.000 description 3
- 108050007209 Nucleobindin-2 Proteins 0.000 description 3
- 239000002202 Polyethylene glycol Substances 0.000 description 3
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 238000007792 addition Methods 0.000 description 3
- 210000004102 animal cell Anatomy 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- 238000004422 calculation algorithm Methods 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 230000004927 fusion Effects 0.000 description 3
- 230000003834 intracellular effect Effects 0.000 description 3
- 239000011159 matrix material Substances 0.000 description 3
- 230000035772 mutation Effects 0.000 description 3
- APVPOHHVBBYQAV-UHFFFAOYSA-N n-(4-aminophenyl)sulfonyloctadecanamide Chemical compound CCCCCCCCCCCCCCCCCC(=O)NS(=O)(=O)C1=CC=C(N)C=C1 APVPOHHVBBYQAV-UHFFFAOYSA-N 0.000 description 3
- 229920001223 polyethylene glycol Polymers 0.000 description 3
- 239000013641 positive control Substances 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- 108010001535 sulfhydryl oxidase Proteins 0.000 description 3
- 238000011144 upstream manufacturing Methods 0.000 description 3
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 2
- 108091033409 CRISPR Proteins 0.000 description 2
- 108020004705 Codon Proteins 0.000 description 2
- 241000699802 Cricetulus griseus Species 0.000 description 2
- RGHNJXZEOKUKBD-SQOUGZDYSA-N D-gluconic acid Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C(O)=O RGHNJXZEOKUKBD-SQOUGZDYSA-N 0.000 description 2
- ZRALSGWEFCBTJO-UHFFFAOYSA-N Guanidine Chemical compound NC(N)=N ZRALSGWEFCBTJO-UHFFFAOYSA-N 0.000 description 2
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 0.000 description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- 101710087172 Procollagen C-endopeptidase enhancer 1 Proteins 0.000 description 2
- 102100041026 Procollagen C-endopeptidase enhancer 1 Human genes 0.000 description 2
- 102000006010 Protein Disulfide-Isomerase Human genes 0.000 description 2
- LCTONWCANYUPML-UHFFFAOYSA-N Pyruvic acid Chemical compound CC(=O)C(O)=O LCTONWCANYUPML-UHFFFAOYSA-N 0.000 description 2
- 108091027544 Subgenomic mRNA Proteins 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 150000007513 acids Chemical class 0.000 description 2
- 238000001042 affinity chromatography Methods 0.000 description 2
- 238000005571 anion exchange chromatography Methods 0.000 description 2
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 2
- 230000008033 biological extinction Effects 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 210000000349 chromosome Anatomy 0.000 description 2
- 230000009089 cytolysis Effects 0.000 description 2
- 235000014113 dietary fatty acids Nutrition 0.000 description 2
- 238000004520 electroporation Methods 0.000 description 2
- 239000003623 enhancer Substances 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- 239000000194 fatty acid Substances 0.000 description 2
- 229930195729 fatty acid Natural products 0.000 description 2
- 150000004665 fatty acids Chemical class 0.000 description 2
- 102000034287 fluorescent proteins Human genes 0.000 description 2
- 108091006047 fluorescent proteins Proteins 0.000 description 2
- IPCSVZSSVZVIGE-UHFFFAOYSA-N hexadecanoic acid Chemical compound CCCCCCCCCCCCCCCC(O)=O IPCSVZSSVZVIGE-UHFFFAOYSA-N 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 210000004897 n-terminal region Anatomy 0.000 description 2
- 239000013642 negative control Substances 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 239000001301 oxygen Substances 0.000 description 2
- 229910052760 oxygen Inorganic materials 0.000 description 2
- 239000000049 pigment Substances 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 108020003519 protein disulfide isomerase Proteins 0.000 description 2
- 230000017854 proteolysis Effects 0.000 description 2
- 238000003259 recombinant expression Methods 0.000 description 2
- 238000005215 recombination Methods 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- 235000000346 sugar Nutrition 0.000 description 2
- 238000013519 translation Methods 0.000 description 2
- 230000035899 viability Effects 0.000 description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 description 1
- NVKAWKQGWWIWPM-ABEVXSGRSA-N 17-β-hydroxy-5-α-Androstan-3-one Chemical compound C1C(=O)CC[C@]2(C)[C@H]3CC[C@](C)([C@H](CC4)O)[C@@H]4[C@@H]3CC[C@H]21 NVKAWKQGWWIWPM-ABEVXSGRSA-N 0.000 description 1
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 1
- ATRRKUHOCOJYRX-UHFFFAOYSA-N Ammonium bicarbonate Chemical compound [NH4+].OC([O-])=O ATRRKUHOCOJYRX-UHFFFAOYSA-N 0.000 description 1
- 239000004254 Ammonium phosphate Substances 0.000 description 1
- 238000010354 CRISPR gene editing Methods 0.000 description 1
- 238000010356 CRISPR-Cas9 genome editing Methods 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- 101100348617 Candida albicans (strain SC5314 / ATCC MYA-2876) NIK1 gene Proteins 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 102100035882 Catalase Human genes 0.000 description 1
- 102000003780 Clusterin Human genes 0.000 description 1
- 108090000197 Clusterin Proteins 0.000 description 1
- 108091026890 Coding region Proteins 0.000 description 1
- 108010035532 Collagen Proteins 0.000 description 1
- 102000008186 Collagen Human genes 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- RGHNJXZEOKUKBD-UHFFFAOYSA-N D-gluconic acid Natural products OCC(O)C(O)C(O)C(O)C(O)=O RGHNJXZEOKUKBD-UHFFFAOYSA-N 0.000 description 1
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N DMSO Substances CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 1
- 102000053602 DNA Human genes 0.000 description 1
- 230000033616 DNA repair Effects 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- 229930091371 Fructose Natural products 0.000 description 1
- 239000005715 Fructose Substances 0.000 description 1
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 244000068988 Glycine max Species 0.000 description 1
- 235000010469 Glycine max Nutrition 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 102000004195 Isomerases Human genes 0.000 description 1
- 108090000769 Isomerases Proteins 0.000 description 1
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 1
- 102000004856 Lectins Human genes 0.000 description 1
- 108090001090 Lectins Proteins 0.000 description 1
- 102000003960 Ligases Human genes 0.000 description 1
- 108090000364 Ligases Proteins 0.000 description 1
- OYHQOLUKZRVURQ-HZJYTTRNSA-N Linoleic acid Chemical compound CCCCC\C=C/C\C=C/CCCCCCCC(O)=O OYHQOLUKZRVURQ-HZJYTTRNSA-N 0.000 description 1
- WHXSMMKQMYFTQS-UHFFFAOYSA-N Lithium Chemical compound [Li] WHXSMMKQMYFTQS-UHFFFAOYSA-N 0.000 description 1
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- GXCLVBGFBYZDAG-UHFFFAOYSA-N N-[2-(1H-indol-3-yl)ethyl]-N-methylprop-2-en-1-amine Chemical compound CN(CCC1=CNC2=C1C=CC=C2)CC=C GXCLVBGFBYZDAG-UHFFFAOYSA-N 0.000 description 1
- CHJJGSNFBQVOTG-UHFFFAOYSA-N N-methyl-guanidine Natural products CNC(N)=N CHJJGSNFBQVOTG-UHFFFAOYSA-N 0.000 description 1
- 102000008730 Nestin Human genes 0.000 description 1
- 108010088225 Nestin Proteins 0.000 description 1
- 102100037369 Nidogen-1 Human genes 0.000 description 1
- 108090000854 Oxidoreductases Proteins 0.000 description 1
- 102000004316 Oxidoreductases Human genes 0.000 description 1
- 235000021314 Palmitic acid Nutrition 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- 102100035846 Pigment epithelium-derived factor Human genes 0.000 description 1
- 108010001014 Plasminogen Activators Proteins 0.000 description 1
- 102000001938 Plasminogen Activators Human genes 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 101100007329 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) COS1 gene Proteins 0.000 description 1
- 101100221606 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) COS7 gene Proteins 0.000 description 1
- 241000256248 Spodoptera Species 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 235000021355 Stearic acid Nutrition 0.000 description 1
- 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 1
- 229930006000 Sucrose Natural products 0.000 description 1
- 235000019486 Sunflower oil Nutrition 0.000 description 1
- 108700029229 Transcriptional Regulatory Elements Proteins 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 240000008042 Zea mays Species 0.000 description 1
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 1
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 1
- 150000001298 alcohols Chemical class 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 239000001099 ammonium carbonate Substances 0.000 description 1
- 235000012501 ammonium carbonate Nutrition 0.000 description 1
- 235000019270 ammonium chloride Nutrition 0.000 description 1
- 229910000148 ammonium phosphate Inorganic materials 0.000 description 1
- 235000019289 ammonium phosphates Nutrition 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 1
- 235000011130 ammonium sulphate Nutrition 0.000 description 1
- PRORZGWHZXZQMV-UHFFFAOYSA-N azane;nitric acid Chemical compound N.O[N+]([O-])=O PRORZGWHZXZQMV-UHFFFAOYSA-N 0.000 description 1
- 150000007514 bases Chemical class 0.000 description 1
- 238000010923 batch production Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- GUBGYTABKSRVRQ-QUYVBRFLSA-N beta-maltose Chemical compound OC[C@H]1O[C@H](O[C@H]2[C@H](O)[C@@H](O)[C@H](O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@@H]1O GUBGYTABKSRVRQ-QUYVBRFLSA-N 0.000 description 1
- 230000027455 binding Effects 0.000 description 1
- 230000015572 biosynthetic process 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
- 238000004364 calculation method Methods 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 101150038500 cas9 gene Proteins 0.000 description 1
- 239000004359 castor oil Substances 0.000 description 1
- 235000019438 castor oil Nutrition 0.000 description 1
- 238000005277 cation exchange chromatography Methods 0.000 description 1
- 125000002091 cationic group Chemical group 0.000 description 1
- 150000001768 cations Chemical class 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000013611 chromosomal DNA Substances 0.000 description 1
- 239000007979 citrate buffer Substances 0.000 description 1
- 239000003240 coconut oil Substances 0.000 description 1
- 235000019864 coconut oil Nutrition 0.000 description 1
- 229920001436 collagen Polymers 0.000 description 1
- 238000004590 computer program Methods 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 235000005822 corn Nutrition 0.000 description 1
- 239000013601 cosmid vector Substances 0.000 description 1
- 238000002425 crystallisation Methods 0.000 description 1
- 230000008025 crystallization Effects 0.000 description 1
- 238000012136 culture method Methods 0.000 description 1
- 235000018417 cysteine Nutrition 0.000 description 1
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- MNNHAPBLZZVQHP-UHFFFAOYSA-N diammonium hydrogen phosphate Chemical compound [NH4+].[NH4+].OP([O-])([O-])=O MNNHAPBLZZVQHP-UHFFFAOYSA-N 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- SWSQBOPZIKWTGO-UHFFFAOYSA-N dimethylaminoamidine Natural products CN(C)C(N)=N SWSQBOPZIKWTGO-UHFFFAOYSA-N 0.000 description 1
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000009977 dual effect Effects 0.000 description 1
- 235000014103 egg white Nutrition 0.000 description 1
- 210000000969 egg white Anatomy 0.000 description 1
- 239000012149 elution buffer Substances 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 210000003527 eukaryotic cell Anatomy 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 238000001943 fluorescence-activated cell sorting Methods 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 239000000174 gluconic acid Substances 0.000 description 1
- 235000012208 gluconic acid Nutrition 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- ZEMPKEQAKRGZGQ-XOQCFJPHSA-N glycerol triricinoleate Natural products CCCCCC[C@@H](O)CC=CCCCCCCCC(=O)OC[C@@H](COC(=O)CCCCCCCC=CC[C@@H](O)CCCCCC)OC(=O)CCCCCCCC=CC[C@H](O)CCCCCC ZEMPKEQAKRGZGQ-XOQCFJPHSA-N 0.000 description 1
- 150000002334 glycols Chemical class 0.000 description 1
- 230000006801 homologous recombination Effects 0.000 description 1
- 238000002744 homologous recombination Methods 0.000 description 1
- 238000004191 hydrophobic interaction chromatography Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 150000002484 inorganic compounds Chemical class 0.000 description 1
- 229910010272 inorganic material Inorganic materials 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 229910000358 iron sulfate Inorganic materials 0.000 description 1
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 239000002523 lectin Substances 0.000 description 1
- 235000020778 linoleic acid Nutrition 0.000 description 1
- OYHQOLUKZRVURQ-IXWMQOLASA-N linoleic acid Natural products CCCCC\C=C/C\C=C\CCCCCCCC(O)=O OYHQOLUKZRVURQ-IXWMQOLASA-N 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 229910052744 lithium Inorganic materials 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 235000019341 magnesium sulphate Nutrition 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 238000000520 microinjection Methods 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 235000019796 monopotassium phosphate Nutrition 0.000 description 1
- WQEPLUUGTLDZJY-UHFFFAOYSA-N n-Pentadecanoic acid Natural products CCCCCCCCCCCCCCC(O)=O WQEPLUUGTLDZJY-UHFFFAOYSA-N 0.000 description 1
- 210000005055 nestin Anatomy 0.000 description 1
- 108010008217 nidogen Proteins 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- QIQXTHQIDYTFRH-UHFFFAOYSA-N octadecanoic acid Chemical compound CCCCCCCCCCCCCCCCCC(O)=O QIQXTHQIDYTFRH-UHFFFAOYSA-N 0.000 description 1
- OQCDKBAXFALNLD-UHFFFAOYSA-N octadecanoic acid Natural products CCCCCCCC(C)CCCCCCCCC(O)=O OQCDKBAXFALNLD-UHFFFAOYSA-N 0.000 description 1
- 235000014593 oils and fats Nutrition 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 235000005985 organic acids Nutrition 0.000 description 1
- 210000001672 ovary Anatomy 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- 108090000102 pigment epithelium-derived factor Proteins 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 239000013600 plasmid vector Substances 0.000 description 1
- 229940127126 plasminogen activator Drugs 0.000 description 1
- 230000008488 polyadenylation Effects 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 239000012562 protein A resin Substances 0.000 description 1
- 235000004252 protein component Nutrition 0.000 description 1
- 230000012846 protein folding Effects 0.000 description 1
- 229940107700 pyruvic acid Drugs 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 238000004007 reversed phase HPLC Methods 0.000 description 1
- 230000037432 silent mutation Effects 0.000 description 1
- 238000001542 size-exclusion chromatography Methods 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000003549 soybean oil Substances 0.000 description 1
- 235000012424 soybean oil Nutrition 0.000 description 1
- 230000009870 specific binding Effects 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 239000008117 stearic acid Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- 239000002600 sunflower oil Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 230000005030 transcription termination Effects 0.000 description 1
- 230000001131 transforming effect 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
- 239000012588 trypsin Substances 0.000 description 1
- 108010087967 type I signal peptidase Proteins 0.000 description 1
- 238000004704 ultra performance liquid chromatography Methods 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
- 241001515965 unidentified phage Species 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
- 239000012138 yeast extract Substances 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
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4728—Calcium binding proteins, e.g. calmodulin
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
- C07K14/43595—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from coelenteratae, e.g. medusae
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4702—Regulators; Modulating activity
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4747—Apoptosis related proteins
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/52—Cytokines; Lymphokines; Interferons
- C07K14/521—Chemokines
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/52—Cytokines; Lymphokines; Interferons
- C07K14/521—Chemokines
- C07K14/523—Beta-chemokines, e.g. RANTES, I-309/TCA-3, MIP-1alpha, MIP-1beta/ACT-2/LD78/SCIF, MCP-1/MCAF, MCP-2, MCP-3, LDCF-1, LDCF-2
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/81—Protease inhibitors
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/81—Protease inhibitors
- C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
- C07K14/811—Serine protease (E.C. 3.4.21) inhibitors
- C07K14/8121—Serpins
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
- C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
-
- 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
-
- 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
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
-
- 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
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
- C12N15/90—Stable introduction of foreign DNA into chromosome
- C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
- C12N15/907—Stable introduction of foreign DNA into chromosome using homologous recombination in mammalian cells
-
- 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
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
-
- 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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0051—Oxidoreductases (1.) acting on a sulfur group of donors (1.8)
-
- 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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- C12N9/18—Carboxylic ester hydrolases (3.1.1)
-
- 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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- C12N9/18—Carboxylic ester hydrolases (3.1.1)
- C12N9/20—Triglyceride splitting, e.g. by means of lipase
-
- 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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
- C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
- C12N9/6472—Cysteine endopeptidases (3.4.22)
-
- 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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
- C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
- C12N9/6489—Metalloendopeptidases (3.4.24)
-
- 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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/90—Isomerases (5.)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y108/00—Oxidoreductases acting on sulfur groups as donors (1.8)
- C12Y108/03—Oxidoreductases acting on sulfur groups as donors (1.8) with oxygen as acceptor (1.8.3)
- C12Y108/03002—Thiol oxidase (1.8.3.2)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y301/00—Hydrolases acting on ester bonds (3.1)
- C12Y301/01—Carboxylic ester hydrolases (3.1.1)
- C12Y301/01034—Lipoprotein lipase (3.1.1.34)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
- C12Y304/22—Cysteine endopeptidases (3.4.22)
- C12Y304/22001—Cathepsin B (3.4.22.1)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y503/00—Intramolecular oxidoreductases (5.3)
- C12Y503/04—Intramolecular oxidoreductases (5.3) transposing S-S bonds (5.3.4)
- C12Y503/04001—Protein disulfide-isomerase (5.3.4.1), i.e. disufide bond-forming enzyme
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/10—Immunoglobulins specific features characterized by their source of isolation or production
- C07K2317/14—Specific host cells or culture conditions, e.g. components, pH or temperature
-
- 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
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- General Engineering & Computer Science (AREA)
- Medicinal Chemistry (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Microbiology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Gastroenterology & Hepatology (AREA)
- Toxicology (AREA)
- Plant Pathology (AREA)
- Physics & Mathematics (AREA)
- Immunology (AREA)
- Cell Biology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Mycology (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Peptides Or Proteins (AREA)
Abstract
本發明關於源自CHO細胞之蛋白分泌因子、表現匣(其中編碼該蛋白分泌因子之核酸序列和編碼靶蛋白之基因係可操作地連接)、包含該表現匣之表現載體、引入該表現載體之轉形細胞及使用該轉形細胞以產生靶蛋白之方法。
Description
本發明關於源自CHO細胞之蛋白分泌因子、表現匣(其中編碼該蛋白分泌因子之核酸序列和編碼靶蛋白之基因係可操作地連接)、包含該表現匣之表現載體、引入該表現載體之轉形細胞及使用該轉形細胞以產生靶蛋白之方法。
重組蛋白可透過用於難以在體內取得之有用的蛋白質組分之基因重組技術,利用微生物或動物細胞系統來大規模製造。重組蛋白可經調節,從而使其可在細胞中表現或分泌到細胞外。然而,細胞內表現之缺點在於蛋白質經常以不可溶之團塊形式積累,且由於難以分離和純化,因而使產製率降低。另一方面,具有正確之蛋白質折疊 的可溶性蛋白質可輕易透過細胞外分泌獲得。因此,就蛋白質產量和質量控制而言,優化之重組蛋白質表現系統對更合適之細胞外分泌很重要。
為了產生重組蛋白,基本上需要諸如宿主細胞、所欲基因、表現載體、選擇標記、啟動子和信號肽序列之組成分。該重組蛋白之品質和產製率將根據這些組成分之選擇而變化。
在信號肽之情況中,其係位於欲製造之重組蛋白的N端區且因此涉及該重組蛋白之表現水準,為允許細胞外分泌之組成分。根據所使用之信號肽可觀察到表現水準之差異,且由於該信號肽之錯誤裂解,該信號肽序列可能保留在該蛋白質之N端,影響該重組蛋白之品質。
因此,選擇不會引起錯誤裂解並可誘導高度表現之信號肽是很重要的。
同時,習知信號肽大多使用源自人類之信號肽,並主要使用CHO細胞作為表現宿主細胞。宿主細胞之間的信號肽可組合使用,但就品質而言可能會引起問題。
在這些情況下,本發明者已做出巨大努力以增加CHO細胞中之表現水準並解決該錯誤裂解問題。結果,本發明者已研發出新穎信號肽,該信號肽為由源自CHO細胞之17至31個胺基酸序列所組成之多肽,本發明者並已藉由確認該信號肽之表現可顯著增加且100%在裂解位點裂解以防止錯誤裂解來完成本發明。
[技術問題]
本發明之目的係提供蛋白分泌因子,該蛋白分泌因子係由SEQ ID NO:1、SEQ ID NO:2或SEQ ID NO:3之胺基酸序列所組成。
本發明之另一目的係提供表現匣,該表現匣中編碼蛋白分泌因子之核酸序列和編碼靶蛋白之基因係可操作地連接,該蛋白分泌因子係由SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4或SEQ ID NO:5之胺基酸序列所組成。
本發明還有另一目的為提供用於分泌靶蛋白之表現載體,該表現載體包含表現匣,該表現匣中編碼蛋白分泌因子之核酸序列和編碼靶蛋白之基因係可操作地連接,該蛋白分泌因子係由SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4或SEQ ID NO:5之胺基酸序列所組成。
本發明還有另一目的為提供轉形細胞,其中該表現載體被引入宿主細胞中。
本發明還有另一目的為提供用於產生靶蛋白之方法,其包含:
i) 培養用於分泌靶蛋白之包含表現載體的轉形細胞,該表現載體包含表現匣,該表現匣中編碼蛋白分泌因子之核酸序列和編碼靶蛋白之基因係可操作地連接,該蛋白分泌因子係由SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4或SEQ ID NO:5之胺基酸序列所組成;和
ii)從該培養細胞之培養基或培養上清液回收該靶蛋白。
[技術解決方案]
本發明將詳細描述於下文中。同時,本文所揭示之每個解釋和示例性實施態樣可應用在其他解釋和示例性實施態樣。亦即,本文所揭示之各種因素的所有組合均屬於本發明之範圍。此外,本發明之範圍不應受下文中提供之具體揭示內容限制。
另外,本技藝之一般技術人士僅使用常規實驗就可能識別或確認許多本文所描述之本發明之特定態樣的同等物。此外,本發明亦意圖包括這些同等物。
為了實現上述目的,本發明之一種態樣係提供源自CHO細胞之新穎蛋白分泌因子。具體而言,本發明提供由SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:9或SEQ ID NO:10之胺基酸序列所組成的蛋白分泌因子。更具體地說,該蛋白分泌因子可由SEQ ID NO:1、SEQ ID NO:2或SEQ ID NO:3之胺基酸序列所組成,但不限於此。
本發明之“蛋白分泌因子”係指與靶蛋白連接以誘導靶蛋白之細胞外分泌之因子且其可由多肽組成。該蛋白分泌因子可促進靶蛋白(即,內源蛋白質和/或外來蛋白質)分泌,且特別地,該蛋白分泌因子可促進抗體之輕鏈和/或重鏈的細胞外分泌,但並不限於此。
本發明中蛋白分泌因子可與“信號序列”或“信號肽(SP)”互換使用。
本發明之蛋白分泌因子可具有SEQ ID NO:1、SEQ ID NO:2或SEQ ID NO:3之胺基酸序列,但不限於此。另外,本發明之蛋白分泌因子可進一步包括由SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:9或SEQ ID NO:10之胺基酸序列所組成的蛋白分泌因子,但不限於此。
於本發明之一具體實施態樣中,該蛋白分泌因子可源自CHO細胞,但不限於此。如本文所使用之術語“CHO細胞”為中國倉鼠卵巢細胞,且可為本技藝常用之用於轉形的宿主細胞。此外,可選擇源自CHO細胞之蛋白分泌因子以增加為宿主細胞之CHO細胞中的表現水準。
本發明中,該由SEQ ID NO:1之胺基酸序列所組成之蛋白分泌因子可為組織蛋白酶B(cathepsin B)(Cat)且可與本發明中之Cat分泌序列互換使用。該由SEQ ID NO:2之胺基酸序列所組成之蛋白分泌因子可為C-C基序趨化因子(CC)且在本發明中可與CC分泌序列互換使用。該由SEQ ID NO:3之胺基酸序列所組成之蛋白分泌因子可為核連蛋白-2(nucleobindin-2)(NUC),且可與本發明中之NUC分泌序列互換使用。
此外,本發明之由SEQ ID NO:4之胺基酸序列所組成的蛋白分泌因子可為簇集蛋白(clusterin)(Clus)且在本發明中可與Clus分泌序列互換使用。該由SEQ ID NO:5之胺基酸序列所組成的蛋白分泌因子可為源自色素上皮之因子(Pig)且在本發明中可與Pig分泌序列互換使用。該由SEQ ID NO:6之胺基酸序列所組成的蛋白分泌因子可為前膠原蛋白C-內肽酶增強子1(Proco)且在本發明中可與Proco分泌序列互換使用。該由SEQ ID NO:7之胺基酸序列所組成的蛋白分泌因子可為巰基氧化酶(Sulf)且在本發明中可與Sulf分泌序列互換使用。該由SEQ ID NO:8之胺基酸序列所組成的蛋白分泌因子可為脂蛋白脂肪酶(Lip)且在本發明中可與Lip分泌序列互換使用。該由SEQ ID NO:9之胺基酸序列所組成的蛋白分泌因子可為巢蛋白-1(nidogen-1)(Nid)且在本發明中可與Nid分泌序列互換使用。該由SEQ ID NO:10之胺基酸序列所組成的蛋白分泌因子可為蛋白質二硫鍵異構酶(Pro)且在本發明中可與Pro分泌序列互換使用。
該編碼由SEQ ID NO:1之胺基酸序列所組成之組織蛋白酶B信號肽的核酸序列可為SEQ ID NO:11之多核苷酸序列,該編碼由SEQ ID NO:2之胺基酸序列所組成之C-C基序趨化因子信號肽的核酸序列可為SEQ ID NO:12之多核苷酸序列,而該編碼由SEQ ID NO:3之胺基酸序列所組成之核連蛋白-2信號肽的核酸序列可為SEQ ID NO:13之多核苷酸序列。
另外,該編碼由SEQ ID NO:4之胺基酸序列所組成之簇集蛋白信號肽的核酸序列可為SEQ ID NO:14之多核苷酸序列,該編碼由SEQ ID NO:5之胺基酸序列所組成之源自色素上皮之因子(Pig)信號肽的核酸序列可為SEQ ID NO:15之多核苷酸序列,該編碼由SEQ ID NO:6之胺基酸序列所組成之前膠原蛋白C-內肽酶增強子1(Proco)信號肽的核酸序列可為SEQ ID NO:16之多核苷酸序列,該編碼由SEQ ID NO:7之胺基酸序列所組成之巰基氧化酶(Sulf)信號肽的核酸序列可為SEQ ID NO:17之多核苷酸序列,該編碼由SEQ ID NO:8之胺基酸序列所組成之脂蛋白脂肪酶(Lip)信號肽的核酸序列可為SEQ ID NO:18之多核苷酸序列,該編碼由SEQ ID NO:9之胺基酸序列所組成之巢蛋白-1(Nid)信號肽的核酸序列可為SEQ ID NO:19之多核苷酸序列,而該編碼由SEQ ID NO:10之胺基酸序列所組成之二硫鍵異構酶(Pro)信號肽的核酸序列可為SEQ ID NO:20之多核苷酸序列。
雖然本發明之蛋白分泌因子被描述為“由具體指定之胺基酸序列所組成之分泌因子”,顯而易見的是,不能排除可能由於在該胺基酸序列上游或下游添加無意義序列而發生的突變、可能自然發生之突變或其沉默突變,只要該分泌因子之活性同等於或對應於該具有對應序列編號之胺基酸序列所組成之分泌因子的活性。即使當該序列存有添加或突變時,其亦在本發明之範圍內。
例如,只要該分泌因子作為信號肽之功能等同於或對應於由上述多核苷酸所組成之核酸分子、顯示出與上述序列具有85%或更高(具體地說,90%或更高,更具體地說95%或更高,再更具體地說98%或更高,或者再更具體地說99%或更高)之同源性和/或同一性的核酸序列,則其亦可不受限制地包括在本發明中。此外,顯而易見的是,序列之一部分具有缺失、修飾、取代或添加的核酸序列亦可包括在本發明之範圍內,只要該核酸序列具有該等同源性。
如本文所使用之術語“同源性”或“同一性”係指介於二個指定之胺基酸序列或核酸序列之間的相關程度,並可以百分比表示。術語“同源性”和“同一性”通常可彼此互換使用。
該保守之多核苷酸或多肽序列的序列同源性或同一性可藉由標準比對算法測定並可與藉由所使用之程式建立的預設空隙懲罰一起使用。基本上,同源序列或同一序列通常被預期在中等或高度嚴格之條件下可與該序列之全部長度或至少約50%、60%、70%、80% 或90%或更多之序列雜交。含有簡併密碼子而非雜交多肽中之密碼子的多核苷酸亦在考量內。
任何二個多核苷酸或多肽序列是否具有同源性、相似性或同一性可使用預設參數,藉由已知之電腦算法,諸如“FASTA”程式(Pearsonet al
., (1988) [Proc. Natl. Acad. Sci
. USA 85]: 2444)測定。或者,可藉由Needleman-Wunsch算法(Needleman and Wunsch, 1970,J. Mol. Biol
. 48: 443-453,其使用EMBOSS程式包之Needleman程式執行(EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000,Trends Genet
. 16: 276-277)(較佳為版本5.0.0或之後的版本)(GCG程式包(Devereux, J.,et al
.,Nucleic Acids Research
12: 387(1984))、BLASTP、BLASTN、FASTA(Atschul, [S.] [F.,] [ET AL
.,J MOLEC BIOL
215]: 403 (1990); Guide to Huge Computers, Martin J. Bishop, [ED.,]Academic Press
, San Diego, 1994,和[CARILLO ETA/.](1988)SIAM J Applied Math
48: 1073)測定。例如,該同源性、相似性或同一性可使用BLAST或國家生物技術信息中心(NCBI)之ClustalW測定。
多核苷酸或多肽之同源性、相似性或同一性可藉由使用,例如GAP電腦程式,諸如Needlemanet al
. (1970),J Mol Biol
. 48: 443(如Smith and Waterman,Adv. Appl. Math
(1981) 2:482中所揭示者)比較序列信息來測定。綜上所述,GAP程式將同源性、相似性或同一性定義為藉由將二個序列中相似比對之符號(即,核苷酸或胺基酸)的數目除以較短者之符號總數所獲得的值。GAP程式之預設參數可包括(1)一元比較矩陣(含有用於同一性之數值1;用於非同一性之數值0);及Gribskovet al
. (1986),Nucl. Acids Res.
14:6745之加權比較矩陣(如Schwartz and Dayhoff, eds.,Atlas of Protein Sequence and Structure
, National Biomedical Research Foundation, pp. 353-358 (1979)中所揭示者)(或EDNAFULL替代矩陣(NCBI NUC4.4之EMBOSS版本));(2)每個間隙罰分為3.0且每個間隙的每個符號另加0.10罰分(或間隙開放罰分為10和間隙延伸罰分為0.5);及(3)終端間隙無罰分。因此,如本文所使用之術語“同源性”或“同一性”係指序列之間的相關性。
本發明之另一態樣提供表現匣,其中編碼蛋白分泌因子之核酸序列和編碼靶蛋白之基因係可操作地連接,該蛋白分泌因子係由SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4或SEQ ID NO:5之胺基酸序列所組成。
本發明之“蛋白分泌因子”如上述。
如本文所使用之術語“靶蛋白”可指內源性表現在宿主細胞中之蛋白質或由被導入宿主細胞中之外源基因表現的蛋白質。該靶蛋白之類型並無特別限制,只要本發明之信號肽序列可增加其細胞外分泌效率即可。
該靶蛋白可為抗體、抗體片段(Fab或ScFv)、融合蛋白、蛋白支架、人生長激素、血清蛋白、免疫球蛋白、細胞因子、α、β或γ干擾素、粒細胞-巨噬細胞集落刺激因子(GM-CSF)、源自血小板之生長因子(PDGF)、磷脂酶活化蛋白(PLAP)、胰島素、腫瘤壞死因子(TNF)、生長因子、激素、降鈣素(calcitonin)、降鈣素基因相關肽(CGRP)、腦啡肽(enkephalin)、生長調節素(somatomedin)、紅血球生成素(erythropoietin)、下丘腦釋放因子、生長分化因子、細胞黏附蛋白、催乳素(prolactin)、絨毛膜促性腺激素(chorionic gonadotropin)、組織纖溶酶原(plasminogen)活化因子、生長激素釋放肽(GHPR)、胸腺體液因子(THF)、天門冬醯胺酶、精胺酸酶、精胺酸脫胺酶、腺苷脫胺酶、過氧化物歧化酶、內毒素酶、過氧化氫酶(catalase)、胰凝乳蛋白酶(chymotrypsin)、脂肪酶、尿酸酶、腺苷二磷酸酶、酪胺酸酶、膽紅素氧化酶、葡萄糖氧化酶、葡萄糖苷酶、半乳糖苷酶、葡萄糖腦苷脂酶(glucocerebrosidase)或葡萄醣醛酸苷酶(glucuronidase),且具體而言,其可為抗體之重鏈蛋白或輕鏈蛋白,但不限於此。
如本文所使用之術語“可操作地連接”係指介於上述基因序列、啟動子序列和信號肽序列之間的功能性鍵聯以啟動和介導編碼本申請案之蛋白分泌因子的核酸序列及編碼該靶蛋白之基因轉錄。該可操作之鍵聯可使用本技藝已知之基因重組技術製備並可使用本技藝已知之連接酶來製備位點特異性DNA鍵聯,但不限於此。
如本文所使用之術語“表現匣”係指調節一或多個基因及其表現之序列,例如,包括各種順式作用轉錄調節元件之任何組合的核酸序列。本發明之表現匣可進一步包括各種元件,例如本技藝公認為對表現調節而言為必要之核酸序列(諸如啟動子和增強子),以及編碼該蛋白分泌因子和靶蛋白之核酸序列。該調節基因表現之序列(即,調節基因轉錄及其轉錄產物之表現的序列)通常被稱為“調節單位”。大部分之調節單元係位於靶基因之編碼序列的上游,從而使其與該靶基因可操作地連接。另外,該表現匣可包括3'非轉錄區(包括在3'端之聚腺苷酸化位點)。
本發明之表現匣可為多核苷酸之組合,其藉由令該編碼蛋白分泌因子之核酸序列與該編碼靶蛋白之基因可操作地連接來允許宿主細胞中之靶蛋白的胞外分泌和表現,該蛋白分泌因子係由SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4或SEQ ID NO:5之胺基酸序列所組成。
本發明還有另一態樣提供表現匣,其中編碼靶蛋白之基因係與編碼蛋白分泌因子之核酸序列可操作地連接,該編碼蛋白分泌因子之核酸序列係由SEQ ID NO:11至SEQ ID NO:20之胺基酸序列所組成,且
本發明之“蛋白分泌因子”、“靶蛋白”、“可操作地連接”和“表現匣”如上述。
本發明中,該由SEQ ID NO:11之多核苷酸序列編碼之蛋白分泌因子可為組織蛋白酶B(Cat),該由SEQ ID NO:12之多核苷酸序列編碼之蛋白分泌因子可為C-C基序趨化因子(CC),該由SEQ ID NO:13之多核苷酸序列編碼之蛋白分泌因子可為核連蛋白-2(Nuc),該由SEQ ID NO:14之多核苷酸序列編碼之蛋白分泌因子可為簇集蛋白(Clus),該由SEQ ID NO:15之多核苷酸序列編碼之蛋白分泌因子可為源自色素上皮之因子(Pig),該由SEQ ID NO:16之多核苷酸序列編碼之蛋白分泌因子可為前膠原蛋白C-內肽酶增強子1(Proco),該由SEQ ID NO:17之多核苷酸序列編碼之蛋白分泌因子可為巰基氧化酶(Sulf),該由SEQ ID NO:18之多核苷酸序列編碼之蛋白分泌因子可為脂蛋白脂肪酶(Lip),該由SEQ ID NO:19之多核苷酸序列編碼之蛋白分泌因子可為巢蛋白-1(Nid)且該由SEQ ID NO:20之多核苷酸序列編碼之蛋白分泌因子可為二硫鍵異構酶(Pro)。
本發明還有另一態樣提供用於分泌靶蛋白之表現載體,該表現載體包含表現匣,該表現匣中編碼蛋白分泌因子之核酸序列和編碼靶蛋白之基因係可操作地連接,該蛋白分泌因子係由SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4或SEQ ID NO:5之胺基酸序列所組成。
本發明之“蛋白分泌因子”、“靶蛋白”、“可操作地連接”和“表現匣”如上述。
如本文所使用之術語“用於分泌靶蛋白之表現載體”係指表現載體,其中該編碼蛋白分泌因子之核酸序列和編碼靶蛋白之基因係可操作地連接,以當該載體被導入宿主細胞中時可誘導靶蛋白之胞外分泌並在該宿主細胞中表現。
如本文所使用之術語“表現載體”通常係指作為載體之雙股DNA片段,該雙股DNA片段中插入編碼靶蛋白之靶DNA片段。本技藝中用於表現蛋白質之表現載體可不受限制地使用。一旦該表現載體在宿主細胞中,該表現載體可不管宿主染色體DNA地複製,且該插入之靶DNA可被表現出。為了增加轉染基因在宿主細胞中之表現水準,該經轉染之基因必須與在所選定之表現宿主細胞中運作的轉錄和轉譯控制序列可操作地連接。
本發明中所使用之表現載體並無特別限制,只要其可在宿主細胞中複製即可,且可使用本技藝中已知之任何載體。常規使用之載體的實例可包括天然或重組質粒、黏粒、病毒和噬菌體。例如,可使用pWE15、M13、λMBL3、λMBL4、λⅨII、λASHII、λAPII、λt10、λt11、Charon4A和Charon21A,等作為噬菌體載體或黏粒載體,並可使用基於pBR、pUC、pBluescriptII、pGEM、pTZ、pCL、pET,等之載體作為質粒載體。具體地說,該載體可為基於pTZ之載體,但不限於此。
於本發明之一具體的實施態樣中,用於分泌靶蛋白之表現載體係基於pTz-D1G1載體(包括韓國專利案編號10-1038126之啟動子的變體),藉由將該編碼蛋白分泌因子之核酸序列與該編碼靶蛋白之基因可操作地連接來製備,該蛋白分泌因子係由SEQ ID NO:1、SEQ ID NO:2或SEQ ID NO:3之胺基酸序列所組成(實施例4)。
該表現載體可進一步包括編碼蛋白分泌因子之核酸序列,該蛋白分泌因子係由SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:9或SEQ ID NO:10之胺基酸序列所組成。
本發明還有另一態樣提供轉形細胞,其中該表現載體被引入宿主細胞中。
本發明之“表現載體”如上述。
如本文所使用之術語“轉形”係指將包括編碼靶多肽之多核苷酸的載體引入宿主細胞中,從而允許由該多核苷酸編碼之蛋白質在宿主細胞中表現之過程。
只要該經轉形之多核苷酸可在宿主細胞中表現,其是否被插入宿主細胞之染色體中且位於其中,或位於該染色體外並不重要,該二種情況均可包括在內。另外,該多核苷酸包括編碼該靶多肽之DNA和RNA。該多核苷酸可以任何形式引入,只要其可被引入宿主細胞並在其中表現即可。例如,可將該多核苷酸以表現匣之形式引入宿主細胞中,該表現匣為包括自我表現所必要之所有元件的基因構建體。該表現匣可常規包括與該多核苷酸可操作地連接之啟動子、轉錄終止信號、核醣體結合結構域和轉譯終止信號。
將本發明之載體轉形的方法包括將核酸引入細胞之任何方法並可藉由根據該宿主細胞選擇本技藝已知之適當標準技術來進行。例如,轉形可經由粒子轟擊(particle bombardment)、電穿孔、磷酸鈣(CaPO4
)沉澱、氯化鈣(CaCl2
)沉澱、顯微注射、聚乙二醇(PEG)技術、DEAE-葡聚醣技術、陽離子脂質體技術、醋酸鋰-DMSO技術進行,但該方法不限於此。
如本文所使用之術語“宿主細胞”係指其中引入核酸分子之真核細胞,該核酸分子具有本發明之蛋白分泌因子的活性並可作為信號肽。該宿主細胞可包括,例如普遍所知之真核宿主,諸如酵母菌;昆蟲細胞,諸如草地貪夜蛾(Spodoptera frupperda);和動物細胞,諸如CHO、COS1、COS7、BSC1、BSC40和BMT10,但不限於此。
本發明中,該宿主細胞之實例可為動物宿主細胞且具體地說,其可為中國倉鼠卵巢細胞(CHO細胞),但不限於此。
於本發明之一具體實施態樣中,使用廣泛用於產生重組蛋白之中國倉鼠卵巢(CHO)細胞來作為宿主細胞(實施例4)。
如本文所使用之術語“轉形體”係指經轉形之動物細胞,其中該包括信號肽和靶蛋白之表現載體被引入作為宿主細胞之CHO細胞中,該信號肽係由SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4或SEQ ID NO:5之胺基酸序列所組成。
於本發明之一具體實施態樣中,該轉形體被確認可增加作為靶蛋白之帕博利珠單抗(pembrolizumab)(即,抗體)之輕鏈和重鏈的表現水準(實施例4)。
本發明還有另一實施態樣提供用於產生靶蛋白之方法,該方法包含:
i) 培養用於分泌靶蛋白之包含表現載體的轉形細胞,該表現載體包含表現匣,該表現匣中編碼蛋白分泌因子之核酸序列和編碼靶蛋白之基因係可操作地連接,該蛋白分泌因子係由SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4或SEQ ID NO:5之胺基酸序列所組成;和
ii)從該培養細胞之培養基或培養上清液回收該靶蛋白。
本發明之“蛋白分泌因子”、“靶蛋白”、“可操作地連接”、“表現匣”、“用於分泌靶蛋白之表現載體”、“宿主細胞”和“轉形體”如上述。
如本文所使用之術語“培養”係指在適當地,人工控制之環境條件下生長該轉形細胞之過程。本發明中,使用CHO細胞作為宿主細胞來產生靶蛋白之方法可使用本技藝中所周知之方法執行。具體而言,該培養可藉由分批過程、分批餵料或以連續方式重複分批餵料之過程進行,但不限於此。
用於培養之培養基應以適當方式滿足特定菌株之要求。可用於本發明之碳源可包括糖和碳水化合物,諸如葡萄糖、蔗糖、乳糖、果糖、麥芽糖、澱粉和纖維素;油類和脂肪類,例如大豆油、葵花油、蓖麻油和椰子油;脂肪酸,諸如棕櫚酸、硬脂酸和亞油酸;醇類,諸如乙醇;和有機酸,諸如葡萄糖酸、醋酸和丙酮酸,但不限於此。這些物質可單獨使用或混合使用。
可用於本發明之氮源可包括蛋白腖、酵母萃取物、肉萃取物、麥芽萃取物、玉米漿、脫脂豆餅和尿素或無機化合物,例如硫酸銨、氯化銨、磷酸銨、碳酸銨和硝酸銨,但不限於此。這些氮源亦可單獨使用或在混合物中使用。
可用於本發明之磷源可包括磷酸二氫鉀或磷酸氫二鉀,或對應之含鈉鹽,但不限於此。另外,該培養基可含有生長所需之金屬鹽,諸如硫酸鎂或硫酸鐵。最後,除上述物質外,可使用必須生長物質,諸如胺基酸和維生素。另外,該培養基中可使用合適之先質。在培養期間,這些物質可以分批或連續方式適當地添加至該培養基中。
可以合適方式將鹼性化合物,諸如氫氧化鈉、氫氧化鉀或氨,或酸性化合物,諸如磷酸或硫酸添加在培養基中以調節該培養基之pH。另外,可使用消泡劑,諸如脂肪酸聚乙二醇酯來抑制氣泡形成。為了將該培養基保持在有氧狀態,可將氧氣或含氧氣體注入該培養基中。該培養基之溫度通常可為20℃至45℃,較佳為25℃至40℃,但可根據條件改變且不限於此。
於本發明之一具體實施態樣中,將該重組表現載體(即,pCB-SP7.2-Pem、pCB-Clus-Pem、pCB-Pig-Pem和pCB-CC-Pem)引入宿主CHO細胞(ExpiCHO-STM
細胞)中並經由分批餵料培養法在30mL之ExpiCHO表現培養基(CHO表現培養基)中培養12天(實施例4)。
本發明之用於產生靶蛋白的方法可包括從該培養基回收靶蛋白之步驟。如本文所使用之術語“回收”為從該培養基獲得靶蛋白之過程並可使用本技藝已知之方法執行,例如離心、過濾、陰離子交換色層分析法、結晶化、HPLC,等,但該方法不限於此。
該回收步驟可包括純化過程且本技藝之技術熟習人士可依需要選擇和利用各種已知之純化過程。例如,可藉由常規色層分析法,諸如免疫親和色層分析法、受體親和力色層分析法、疏水性交互作用色層分析法、凝集素親和力色層分析法、尺寸排阻色層分析法、陽離子或陰離子交換色層分析法、高效液相色層分析法(HPLC)和反相HPLC將該宿主細胞與該宿主細胞之培養基或培養上清液分開。另外,當所欲之蛋白質為具有特異標籤、標記或螯合部分之融合蛋白時,可藉由特異性結合伴侶或藥物純化之。該純化之蛋白質可裂解成所需之蛋白質區,諸如除去分泌因子,或者其可保持原樣。所需之蛋白質形式(包括額外之胺基酸)可藉由在切割過程期間切割該融合蛋白來產生。
本發明之由SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4或SEQ ID NO:5之胺基酸序列所組成的蛋白分泌因子可為在該靶蛋白之N端裂解位點被精確裂解的分泌因子。
該信號肽係位於欲製造之重組蛋白的N端區域且當該靶蛋白轉位時,其被信號肽酶降解。然而,現存之蛋白分泌因子可能時常由於錯誤裂解問題而使靶蛋白之品質降級。該“錯誤裂解”係指其中該信號肽未在正確位置完全降解且該信號肽序列部分保留在該靶蛋白之N端的現象。
於本發明之一具體實施態樣中,該蛋白分泌因子(信號肽)之裂解係使用純化之靶蛋白,藉由Q-TOF MS質譜儀來確認。結果,證實在預測之裂解位點觀察到100%裂解。
因此,包含本發明之蛋白分泌因子(信號肽)的表現載體可透過該重組蛋白之有效表現和分泌來增加靶蛋白之生產率,且可為用於解決該錯誤裂問題之功能強大的遺傳工具。
[有益效果]
本發明之蛋白分泌因子,即,該信號肽可透過高水準表現來顯著增加重組蛋白之生產率且可預期用來作為功能強大的遺傳工具,其可藉由在裂解位點100%裂解來解決常規信號肽之錯誤裂解問題。
實施例1. 源自CHO細胞之新穎信號肽序列的製備方法
1-1. CHO HCP質量分析
如下述,將四種類型(ADH、BSA、PHO和ENL)之MassPREPTM蛋白分解標準物添加在以胰蛋白酶處理的CHO細胞(DXB11)之培養基中。在四種類型之MassPREPTM蛋白分解標準物中,PHO係作為用於計算各宿主細胞蛋白質(HCP)之濃度的內部標準。使用2D LC(高pH RP/低pH RP)-Q-TOF(UDMSe)方法分析每個樣品之宿主細胞蛋白質(HCP)。
藉由將每一分液直接注入Q-TOF MS之2D柱中來獲得每個分液之MS數據(UDMSe)。使用ProteinLynx Global SERVER(PLGS,3.0.2版)軟體將以上述方式獲得之10個分液的MS數據(UDMSe)合併成一個數據。之後,使用每個樣品之合併數據和中國倉鼠蛋白質數據庫鑑定該HCP並使用PHO作為內部標準來計算每個HCP之濃度。
1-2. 從CHO HCP數據選擇信號肽
將蛋白質依高濃度之順序排列,並從CHO基因組數據庫(http://chogenome.org)確認每個蛋白質之胺基酸序列。將由此獲得之胺基酸序列輸入SignalP4.1 Server(http://www.cbs.dtu.dk/services/SignalP/)中以預測是否存有分泌蛋白和信號肽序列(表1、第1圖和表4)。
實施例2. 經由暫時表現選擇源自CHO之高效信號肽
2-1. 用於暫時表現之重組蛋白表現載體的製備方法
為了證實實施例1中所選擇之10種類型的信號肽是否可作為通用之分泌因子,選擇mCherry(pmCherry載體,Clontech公司,632522)蛋白作為靶蛋白。
基於該編碼mCherry蛋白之基因,使用從CHO HCP大量數據鑑別之信號肽序列和含有KpnI/XhoI之引物進行PCR,並構建由該10種類型之信號肽序列表現之mCherry。
當該信號肽之長度長時,將引物分割為二,並進行二次PCR。以KpnI和XhoI裂解含有該10種類型之信號肽序列的mCherry PCR產物,然後將其選殖入pcDNA3.1(+)(Invitrogen,目錄編號V790-20)以構建表現載體。
此外,以相同方式製備與該SP7.2信號肽(韓國專利公開公報10-2015-0125402 A)(其為已知之分泌因子)融合之mCherry蛋白表現載體以用來作為陽性對照組。該SP7.2信號肽之序列顯示於表3中。
2-2. 暫時表現
根據CHO-S細胞株Amaxa 4D-Nucleofector方案來轉染該10種類型之信號肽所表現之各mCherry表現載體(1mL)。之後,在第2天和第6天,測量胞內螢光和該從培養基分泌之螢光蛋白的螢光值(第3圖)。
在胞內螢光之情況中,使用FACS(Accuri)來測量直方圖中高於該陰性對照組(空載體,pMaxGFP)之部分的平均值。
在該分泌入培養基之螢光蛋白之螢光值的情況中,在第2天和第6天採取100 μ0樣品,然後離心以獲得單獨之上清液並使用多重檢測儀在587/610 nm測量螢光。
結果證實在培養基中測得具有高螢光值之信號肽為Cat、CC、Nuc、Clus和Pig。此外,顯示出表現高於陽性對照組SP7.2(SP7.2將作為陽性對照組)的四種分泌因子類型(Clus、Pig、Nuc和CC)和在細胞中具有高螢光值之一種信號肽類型(Proco)被選定作為陰性對照組。
實施例3. 透過位點特異性整合比較表現
3-1. 構建用於位點特異性整合之表現載體
將mCherry序列,包括在實施例2中所選定之5種類型的信號肽(Clus、Pig、Nuc、CC和Proco)和對照組SP7.2一致地插入CHO基因組之特定位點以定量比較該表現水準(第4和5圖)。
該插入位點係設在Hprt位點並參考J.S Leeet al., 2015
, “Site-Specific integration in CHO cells mediated by CRISPR/Cas9 and homology-directed DNA repair pathway”,Sci. Rep.,
5設計同源臂序列和sgRNA序列。
在5'同源臂之情況下,使用含有Bg1Ⅱ和NruI酶切位點之引物及作為模板之CHO-S基因組來進行PCR,然後,將其選殖入以Bg1II/NruI酶切分解之pcDNA3.1(+)載體中。
在3'同源臂之情況下,使用含有SalI位點之各引物及作為模板CHO-S基因組來進行PCR,然後,製造SalI單一切口及插入5'同源臂之載體並將其選殖入NeoR基因之下游(pcDNA3.1_hprt)。
為了確認僅有那些已經歷同源重組者插入基因組中,在5'同源臂之上游區構建Cmy-GFP-BHG pA片段並將其插入SpeI和BG1Ⅱ(pcDNA3 .1_G_hprt)中以用於雙重選擇。
在GFP片段之情況中,首先將其插入具有NcoI/XbaI之pcDNA3.1(+)載體之MCS,然後使用含有SpeI和BglⅡ限制位點之引物進行PCR。之後,利用Bg1II位點將SpeI插入含有該同源性之載體(pcDNA_hprt)中。
使用該完成之pcDNA3.1_G_hprt載體,以KpnI和XhoI切開該含有信號肽序列之mCherry基因序列並將其插入MCS區。
結果,構建基於pcDNA3.1之表現載體,該表現載體含有為CMV-EGFP-pA-5' Hprt同源臂-CMV-信號肽候選物-mCherry-BGH pA-NeoR選擇標記匣-3'Hprt同源臂形式的表現匣。
3-2. 位點特異性整合
利用CRISPR-Cas9執行敲入以將6種類型之用於位點特異性整合的載體插入CHO-S基因組中之Hprt位點。將240 ng之sgRNA、1,250 ng之cas9蛋白和1μg之供體載體獨立地與Nucleofector溶液混合以製備50 μL混合物。
首先將CHO-S 1×106
個細胞溶於50 μL之Nucleofector中,再與該預先製備之混合物混合,隨後,將該100 μL之最終混合物進行電穿孔。
進行電穿孔後,將該混合物與0.5mL之培養基混合,加入2.5 mL之培養基中,並培養在36.5℃和5%CO2
下之6孔盤板中。
2天後,在含有Zeneticin(0.5mg/L)之CD CHO培養基中進行選擇,然後將細胞傳代培養,直至回復90%存活力。
回復90%存活力後,以3×105
細胞/mL之濃度將4 mL之細胞培養在6孔盤中,一式二份並每2至3天測量Vi-細胞和培養基之螢光值(587nm/610 nm)(第5圖)。
結果證實CC、Clus和Pig分泌肽顯示出其表現高於對照組SP7.2之表現。
實施例4. 抗PD-1抗體之表現和質量分析
4-1. 製備用於產生抗PD-1抗體之表現載體。
透過位點特異性整合比較信號肽之表現能力後,將與4種類型之信號肽,包括顯示出高表現之CC、Clus和Pig(CC、Pig、Clus和SP7.2)融合的抗PD-1抗體表現出。使用帕博利珠單抗(Keytruda®)抗體序列作為靶蛋白。
合成對應於該輕鏈和重鏈之胺基酸序列的DNA序列,接著,透過重疊PCR產生與各肽信號序列融合之序列。
在輕鏈之情況中,以BamHI和XhoI將該胺基酸序列限制性切開,在重鏈之情況中,以AscI和NotI將該胺基酸序列限制性切開,然後將抗體插入pTz-D1G1載體(pcDNA3.1(+)之變體)(包括KR專利案10-1038126B1號之啟動子)中。
pCB_SP7.2_Pem
'(N端)-[BamHI限制位-信號肽(SEQ ID NO:33)-Pem輕鏈(SEQ ID NO:58)-XhoI限制位]-(C端)'/'(N端)-[AscI限制位-信號肽(SEQ ID NO:33)-Pem重鏈(SEQ ID NO:59)-NotI限制位]-(C端)'
pCB_Clus_Pem
'(N端)-[BamHI限制位-信號肽(SEQ ID NO:4)-Pem輕鏈(SEQ ID NO:58)-XhoI限制位]-(C端)'/'(N端)-[AscI限制位-信號肽(SEQ ID NO:4)-Pem重鏈(SEQ ID NO:59)-NotI限制性位點]-(C端)'
pCB_CC_Pem
'(N端)-[ BamHI限制位-信號肽(SEQ ID NO:2)-Pem輕鏈(SEQ ID NO:58)-XhoI限制位]-(C端)'/'(N端)-[AscI限制位-信號肽(SEQ ID NO:2)-Pem重鏈(SEQ ID NO:59)-NotI限制位]-(C端)'
pCB_Pig_Pem
'(N端)-[BamHI限制位-信號肽(SEQ ID NO:5)-Pem輕鏈(SEQ ID NO:58)-XhoI限制位]-(C端)'/'(N端)-[AscI限制位-信號肽(SEQ ID NO:5)-Pem重鏈(SEQ ID NO:59)-NotI限制位]-(C端)'
4-2. 抗PD1抗體之表現
將所製備之重組表現載體pCB-SP7.2-Pem、pCB-Clus-Pem、pCB-Pig-Pem和pCB-CC-Pem引入ExpiCHO-STM
細胞(Thermo-Fisher科技)中並培養在ExpiCHO表現基質(Thermo-Fisher科技;30mL)中12天(分批餵料培養;第1天&第5天餵料)以表現該融合多肽(即,帕博利珠單抗)。
4-3. 抗PD1抗體之純化和質量分析
藉由ProteinA純化該透過重組載體表現所產生之融合多肽。具體而言,使用0.22μm過濾器將該回收之培養液過濾,然後將經充填蛋白A樹脂之管柱(Hitrap MSS、GE醫療護理,11-0034-93)安裝在AKTATM
Avant25 (GE醫療護理生命科學)上並使PBS緩衝液通過以平衡該管柱。
將經過濾之培養液注入管柱後,再次令PBS緩衝液通過該管柱以洗滌之。完成洗滌管柱後,令洗提緩衝液(檸檬酸鹽緩衝液,pH 3.5)通過該管柱以將該靶蛋白洗提出。使用Amicon超濾裝置(MWCO 30K,默克公司)和離心機將該洗提液濃縮。進行濃縮後,以PBS進行緩衝液交換。
(*消光係數(0.1%):其為在蛋白質濃度為0.1%(1 g/L)且一級序列上之所有半胱胺酸均被氧化以形成二硫鍵之假設下在280 nm之理論吸光度。經由ProtParam工具(https://web.expasy.org/protparam/)計算
使用純化之靶蛋白,利用Q-TOF MS來確認在該蛋白質N端之信號肽存在錯誤裂解(第6圖)。將濃度稀釋成1mg/mL後,先以PNGaseF,接著以6M胍和DTT處理該蛋白質,然後裝填在Q-TOF MS(RMM-MT-001:ACQUITY UPLC + Q-TOF SYNAPT G2(Waters))上。
結果證實在預測之裂解位點觀察到100%裂解。
基於這些結果,該信號肽(本發明之源自CHO細胞之蛋白分泌因子)藉由增加該重組蛋白之表現水準並透過質量分析確認在預測之裂解位點可觀察到100%裂解來提高生產率,此意味本發明之信號肽可為能夠解決錯誤裂解問題之功能強大的遺傳工具,錯誤裂解為現有之蛋白分泌因子的問題。
儘管本發明已參考特定之說明性實施態樣描述,本發明所屬之技藝的技術熟習人士將理解,在不脫離本發明之技術精神或必要特徵之情況下,本發明可以其他特定形式體現。因此,上述之實施態樣在所有方面均被視為是說明性的而非限制性的。此外,本發明之範圍係由所附之申請專利範圍定義而非由該詳細描述來限定,且應理解的是,從本發明之含義和範圍及其同等物衍生出之所有修飾或變化均包括在所附之申請專利範圍內。
[第1圖]之圖形係使用SignalP4.1來預測該信號肽。
[第2圖]之圖示係透過暫時表現來確認信號肽-mCherry之表現水準。
[第3圖]之圖示顯示用於位點特異性整合的載體圖譜。
[第4圖]之圖示比較該位點特異性整合之細胞中的mCherry之表現水準。
[第5圖]之圖形顯示大量數據,該數據確認與SP7.2和Clus融合之抗PD-1抗體的裂解。
[較佳實施態樣之詳細描述]
下文中將藉由實施例更詳細地描述本發明。然而,給予之這些實施例僅為了說明目的,本發明之範圍並不意圖受限於或被這些實施例限制。
Claims (19)
- 一種蛋白分泌因子,其係由SEQ ID NO:1、SEQ ID NO:2或SEQ ID NO:3之胺基酸序列所組成。
- 如請求項1之蛋白分泌因子,其中該蛋白質為內源蛋白質或外來蛋白質。
- 一種表現匣,其中編碼蛋白分泌因子之核酸序列和編碼靶蛋白之基因係可操作地連接,該蛋白分泌因子係由SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4或SEQ ID NO:5之胺基酸序列所組成。
- 如請求項3之表現匣,其中該靶蛋白係選自由下列所組成之群組:抗體、抗體片段(Fab或ScFv)、融合蛋白、蛋白支架、人生長激素、血清蛋白、免疫球蛋白、細胞因子、α、β或γ干擾素、粒細胞-巨噬細胞集落刺激因子(GM-CSF)、源自血小板之生長因子(PDGF)、磷脂酶活化蛋白(PLAP)、胰島素、腫瘤壞死因子(TNF)、生長因子、激素、降鈣素(calcitonin)、降鈣素基因相關肽(CGRP)、腦啡肽(enkephalin)、生長調節素(somatomedin)、紅血球生成素(erythropoietin)、下丘腦釋放因子、生長分化因子、細胞黏附蛋白、催乳素(prolactin)、絨毛膜促性腺激素(chorionic gonadotropin)、組織纖溶酶原(plasminogen)活化因子、生長激素釋放肽(GHPR)、胸腺體液因子(THF)、天門冬醯胺酶、精胺酸酶、精胺酸脫胺酶、腺苷脫胺酶、過氧化物歧化酶、內毒素酶、過氧化氫酶(catalase)、胰凝乳蛋白酶(chymotrypsin)、脂肪酶、尿酸酶、腺苷二磷酸酶、酪胺酸酶、膽紅素氧化酶、葡萄糖氧化酶、葡萄糖苷酶、半乳糖苷酶、葡萄糖腦苷脂酶(glucocerebrosidase)和葡萄醣醛酸苷酶(glucuronidase)。
- 如請求項3之表現匣,其中該編碼由SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4或SEQ ID NO:5之胺基酸序列所組成之該蛋白分泌因子之核酸序列係由SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:13、SEQ ID NO:14或SEQ ID NO:15之核酸序列所組成。
- 如請求項3之表現匣,其中該表現匣進一步包含核酸序列,該核酸序列編碼由SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:9和SEQ ID NO:10之胺基酸序列所組成之蛋白分泌因子中任一者。
- 如請求項3之表現匣,其中當該靶蛋白在細胞中表現時,該表現匣表現其中編碼該分泌因子之核酸序列已自該表現匣移除之未添加額外之胺基酸的原樣靶蛋白。
- 一種用於分泌靶蛋白之表現載體,其包含表現匣,該表現匣中編碼蛋白分泌因子之核酸序列和編碼靶蛋白之基因係可操作地連接,該蛋白分泌因子係由SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4或SEQ ID NO:5之胺基酸序列所組成。
- 如請求項8之表現載體,其中該靶蛋白係選自由下列所組成之群組:抗體、抗體片段(Fab或ScFv)、融合蛋白、蛋白支架、人生長激素、血清蛋白、免疫球蛋白、細胞因子、α、β或γ干擾素、粒細胞-巨噬細胞集落刺激因子(GM-CSF)、源自血小板之生長因子(PDGF)、磷脂酶活化蛋白(PLAP)、胰島素、腫瘤壞死因子(TNF)、生長因子、激素、降鈣素、降鈣素基因相關肽(CGRP)、腦啡肽、生長調節素、紅血球生成素、下丘腦釋放因子、生長分化因子、細胞黏附蛋白、催乳素、絨毛膜促性腺激素、組織纖溶酶原活化因子、生長激素釋放肽(GHPR)、胸腺體液因子(THF)、天門冬醯胺酶、精胺酸酶、精胺酸脫胺酶、腺苷脫胺酶、過氧化物歧化酶、內毒素酶、過氧化氫酶、胰凝乳蛋白酶、脂肪酶、尿酸酶、腺苷二磷酸酶、酪胺酸酶、膽紅素氧化酶、葡萄糖氧化酶、葡萄糖苷酶、半乳糖苷酶、葡萄糖腦苷脂酶和葡萄醣醛酸苷酶。
- 如請求項8之表現載體,其中編碼由SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4或SEQ ID NO:5之胺基酸序列所組成之該蛋白分泌因子之核酸序列係由SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:13、SEQ ID NO:14或SEQ ID NO:15之核酸序列所組成。
- 如請求項8之表現載體,其中該表現載體進一步包含核酸序列,該核酸序列編碼由SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:9和SEQ ID NO:10之胺基酸序列所組成之蛋白分泌因子中任一者。
- 如請求項8之表現載體,其中當該靶蛋白在細胞中表現時,該表現匣表現其中編碼該分泌因子之核酸序列已自該表現匣移除之未添加額外之胺基酸的原樣靶蛋白。
- 一種轉形細胞,其中如請求項8至12中任一項之表現載體被引入宿主細胞中。
- 如請求項13之轉形細胞,其中該宿主細胞為中國倉鼠卵巢細胞(CHO細胞)。
- 一種用於產生靶蛋白之方法,其包含: i) 培養用於分泌靶蛋白之包含表現載體的轉形細胞,該表現載體包含表現匣,該表現匣中編碼蛋白分泌因子之核酸序列和編碼靶蛋白之基因係可操作地連接,該蛋白分泌因子係由SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4或SEQ ID NO:5之胺基酸序列所組成;和 ii) 從該培養細胞之培養基或培養上清液回收該靶蛋白。
- 如請求項15之方法,其進一步包含純化該回收之靶蛋白。
- 如請求項15之方法,其中該宿主細胞為中國倉鼠卵巢細胞(CHO細胞)。
- 如請求項15之方法,其中該由SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4或SEQ ID NO:5之胺基酸序列所組成之蛋白分泌因子係在該靶蛋白之N端裂解位點裂解。
- 如請求項15之方法,其中該靶蛋白為其中編碼該分泌因子之核酸序列已經移除之未添加額外之胺基酸的靶蛋白自身。
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
KR20190158447 | 2019-12-02 | ||
KR10-2019-0158447 | 2019-12-02 |
Publications (2)
Publication Number | Publication Date |
---|---|
TW202128735A true TW202128735A (zh) | 2021-08-01 |
TWI841809B TWI841809B (zh) | 2024-05-11 |
Family
ID=
Also Published As
Publication number | Publication date |
---|---|
WO2021112540A1 (ko) | 2021-06-10 |
KR20230115276A (ko) | 2023-08-02 |
EP4063379A4 (en) | 2023-07-26 |
JP2023504648A (ja) | 2023-02-06 |
BR112022010460A2 (pt) | 2022-09-06 |
KR102559048B1 (ko) | 2023-07-26 |
MX2022006522A (es) | 2022-09-12 |
KR20210069001A (ko) | 2021-06-10 |
CN114761418A (zh) | 2022-07-15 |
EP4063379A1 (en) | 2022-09-28 |
US20230234995A1 (en) | 2023-07-27 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US11046975B2 (en) | Bicistronic expression vector for antibody expression and method for producing antibody using same | |
KR101190605B1 (ko) | 활성형 재조합 혈액응고 9인자의 대량생산 방법 | |
JP2021184745A (ja) | 新規の最小utr配列 | |
JP2022516572A (ja) | 合成分子フィードバック回路及びその使用方法 | |
CN112029791A (zh) | 选择真核宿主细胞的选择载体和方法 | |
JP5662363B2 (ja) | 難発現性タンパク質の分泌のためのタンパク質融合因子(tfp)を明らかにする方法、タンパク質融合因子(tfp)ライブラリーを製造する方法、及び難発現性タンパク質の組み換え的生産方法 | |
KR20170132784A (ko) | 글로빈 유전자 클러스터의 조절 요소를 포함하는 진핵생물 발현 벡터 | |
JP4655388B2 (ja) | タンパク質の生産方法 | |
JP6064915B2 (ja) | 発現ベクターおよび蛋白質の製造方法 | |
JP2007519417A (ja) | 形質転換サッカロマイセス・セレビシエ菌株及びそれを使用したlk8蛋白質の生産方法 | |
WO2019030069A2 (en) | USE OF CONSTITUTIVELY ACTIVE GROWTH FACTOR RECEPTOR VARIANTS AS SELECTION MARKERS FOR THE GENERATION OF STABLE PRODUCTION CELL LINES | |
TW202128735A (zh) | 源自cho細胞之蛋白分泌因子和包含彼之表現載體 | |
Li et al. | Expression, purification, and characterization of recombinant human pancreatic duodenal homeobox-1 protein in Pichia pastoris | |
US9476081B2 (en) | Method for producing protein | |
JP2018509904A (ja) | 哺乳動物細胞における組換えタンパク質生産のためのビタミンならびにビタミン代謝関連の遺伝子およびタンパク質の使用 | |
WO2015182792A1 (ja) | ウリジンとn-アセチル-d-マンノサミンとを含有する培地 | |
EP3417054B1 (en) | Cell lines for producing recombinant glycoproteins with di-antennary n-glycans, methods using the same, and recombinant glycoproteins | |
AU2020226714A1 (en) | Cell penetrating transposase | |
EP3732290A1 (en) | Host cells with enhanced protein expression efficiency and uses thereof | |
AU2004282558A1 (en) | FLP-mediated recombination | |
Schloßhauer et al. | Promoting the production of challenging proteins via induced expression in CHO cells and modified cell-free lysates harboring T7 RNA polymerase and mutant eIF2α | |
US9315565B2 (en) | Method for producing protein |