JP4772668B2 - 真核細胞におけるo−グリコシレーションの回避方法 - Google Patents
真核細胞におけるo−グリコシレーションの回避方法 Download PDFInfo
- Publication number
- JP4772668B2 JP4772668B2 JP2006510705A JP2006510705A JP4772668B2 JP 4772668 B2 JP4772668 B2 JP 4772668B2 JP 2006510705 A JP2006510705 A JP 2006510705A JP 2006510705 A JP2006510705 A JP 2006510705A JP 4772668 B2 JP4772668 B2 JP 4772668B2
- Authority
- JP
- Japan
- Prior art keywords
- protein
- amino acid
- glycosylation
- sugar
- threonine
- 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.)
- Expired - Fee Related
Links
- 230000004989 O-glycosylation Effects 0.000 title claims description 50
- 238000000034 method Methods 0.000 title claims description 21
- 210000003527 eukaryotic cell Anatomy 0.000 title description 7
- 108090000623 proteins and genes Proteins 0.000 claims description 99
- 102000004169 proteins and genes Human genes 0.000 claims description 98
- AYFVYJQAPQTCCC-GBXIJSLDSA-N L-threonine Chemical compound C[C@@H](O)[C@H](N)C(O)=O AYFVYJQAPQTCCC-GBXIJSLDSA-N 0.000 claims description 43
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 37
- 150000001413 amino acids Chemical class 0.000 claims description 26
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 24
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 claims description 22
- 239000004473 Threonine Substances 0.000 claims description 22
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 claims description 19
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 claims description 12
- 238000004458 analytical method Methods 0.000 claims description 3
- 235000018102 proteins Nutrition 0.000 description 85
- 230000004048 modification Effects 0.000 description 47
- 238000012986 modification Methods 0.000 description 47
- 125000003275 alpha amino acid group Chemical group 0.000 description 27
- 235000001014 amino acid Nutrition 0.000 description 25
- 229940024606 amino acid Drugs 0.000 description 24
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 description 23
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 22
- 239000002299 complementary DNA Substances 0.000 description 22
- 125000000341 threoninyl group Chemical group [H]OC([H])(C([H])([H])[H])C([H])(N([H])[H])C(*)=O 0.000 description 20
- 125000003607 serino group Chemical group [H]N([H])[C@]([H])(C(=O)[*])C(O[H])([H])[H] 0.000 description 17
- 241000235058 Komagataella pastoris Species 0.000 description 16
- 125000000539 amino acid group Chemical group 0.000 description 15
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 12
- 102000004196 processed proteins & peptides Human genes 0.000 description 11
- 108091035707 Consensus sequence Proteins 0.000 description 10
- 102000016776 Midkine Human genes 0.000 description 10
- 108010092801 Midkine Proteins 0.000 description 10
- 125000004066 1-hydroxyethyl group Chemical group [H]OC([H])([*])C([H])([H])[H] 0.000 description 9
- 239000004471 Glycine Substances 0.000 description 9
- 210000004899 c-terminal region Anatomy 0.000 description 9
- 239000013615 primer Substances 0.000 description 9
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 9
- 230000000694 effects Effects 0.000 description 8
- 102000004190 Enzymes Human genes 0.000 description 7
- 108090000790 Enzymes Proteins 0.000 description 7
- 229940088598 enzyme Drugs 0.000 description 7
- 239000002609 medium Substances 0.000 description 7
- 239000002773 nucleotide Substances 0.000 description 7
- 125000003729 nucleotide group Chemical group 0.000 description 7
- 239000013598 vector Substances 0.000 description 7
- 230000008859 change Effects 0.000 description 6
- 108020004414 DNA Proteins 0.000 description 5
- 238000004440 column chromatography Methods 0.000 description 5
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 4
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 4
- 229920002684 Sepharose Polymers 0.000 description 4
- 239000013604 expression vector Substances 0.000 description 4
- 125000003630 glycyl group Chemical group [H]N([H])C([H])([H])C(*)=O 0.000 description 4
- 238000004519 manufacturing process Methods 0.000 description 4
- 238000004949 mass spectrometry Methods 0.000 description 4
- 108010025188 Alcohol oxidase Proteins 0.000 description 3
- 241000228212 Aspergillus Species 0.000 description 3
- WQZGKKKJIJFFOK-QTVWNMPRSA-N D-mannopyranose Chemical compound OC[C@H]1OC(O)[C@@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-QTVWNMPRSA-N 0.000 description 3
- 108020001019 DNA Primers Proteins 0.000 description 3
- 239000003155 DNA primer Substances 0.000 description 3
- 241000588724 Escherichia coli Species 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 241001506991 Komagataella phaffii GS115 Species 0.000 description 3
- 238000005481 NMR spectroscopy Methods 0.000 description 3
- 241000235648 Pichia Species 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- 235000018417 cysteine Nutrition 0.000 description 3
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 3
- 238000013461 design Methods 0.000 description 3
- 238000004520 electroporation Methods 0.000 description 3
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 3
- 239000013612 plasmid Substances 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 238000004366 reverse phase liquid chromatography Methods 0.000 description 3
- 238000001179 sorption measurement Methods 0.000 description 3
- 238000006467 substitution reaction Methods 0.000 description 3
- 230000001629 suppression Effects 0.000 description 3
- 230000014616 translation Effects 0.000 description 3
- BRZYSWJRSDMWLG-DJWUNRQOSA-N (2r,3r,4r,5r)-2-[(1s,2s,3r,4s,6r)-4,6-diamino-3-[(2s,3r,4r,5s,6r)-3-amino-4,5-dihydroxy-6-[(1r)-1-hydroxyethyl]oxan-2-yl]oxy-2-hydroxycyclohexyl]oxy-5-methyl-4-(methylamino)oxane-3,5-diol Chemical compound O1C[C@@](O)(C)[C@H](NC)[C@@H](O)[C@H]1O[C@@H]1[C@@H](O)[C@H](O[C@@H]2[C@@H]([C@@H](O)[C@H](O)[C@@H]([C@@H](C)O)O2)N)[C@@H](N)C[C@H]1N BRZYSWJRSDMWLG-DJWUNRQOSA-N 0.000 description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 2
- UHPMCKVQTMMPCG-UHFFFAOYSA-N 5,8-dihydroxy-2-methoxy-6-methyl-7-(2-oxopropyl)naphthalene-1,4-dione Chemical compound CC1=C(CC(C)=O)C(O)=C2C(=O)C(OC)=CC(=O)C2=C1O UHPMCKVQTMMPCG-UHFFFAOYSA-N 0.000 description 2
- 108020004705 Codon Proteins 0.000 description 2
- 108020004635 Complementary DNA Proteins 0.000 description 2
- 101900345593 Escherichia coli Alkaline phosphatase Proteins 0.000 description 2
- 241000223218 Fusarium Species 0.000 description 2
- 101000990990 Homo sapiens Midkine Proteins 0.000 description 2
- 102000004856 Lectins Human genes 0.000 description 2
- 108090001090 Lectins Proteins 0.000 description 2
- 241000235395 Mucor Species 0.000 description 2
- 125000001429 N-terminal alpha-amino-acid group Chemical group 0.000 description 2
- 241000221960 Neurospora Species 0.000 description 2
- 239000012506 Sephacryl® Substances 0.000 description 2
- 241000223259 Trichoderma Species 0.000 description 2
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 2
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 2
- 229960000723 ampicillin Drugs 0.000 description 2
- 210000004102 animal cell Anatomy 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- 239000002131 composite material Substances 0.000 description 2
- 239000012228 culture supernatant Substances 0.000 description 2
- 238000010828 elution Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 238000010353 genetic engineering Methods 0.000 description 2
- 102000006602 glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 description 2
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 2
- 102000053521 human MDK Human genes 0.000 description 2
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 2
- 239000002523 lectin Substances 0.000 description 2
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 108091008146 restriction endonucleases Proteins 0.000 description 2
- 230000003248 secreting effect Effects 0.000 description 2
- 230000028327 secretion Effects 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 238000001262 western blot Methods 0.000 description 2
- 239000007222 ypd medium Substances 0.000 description 2
- DQJCDTNMLBYVAY-ZXXIYAEKSA-N (2S,5R,10R,13R)-16-{[(2R,3S,4R,5R)-3-{[(2S,3R,4R,5S,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy}-5-(ethylamino)-6-hydroxy-2-(hydroxymethyl)oxan-4-yl]oxy}-5-(4-aminobutyl)-10-carbamoyl-2,13-dimethyl-4,7,12,15-tetraoxo-3,6,11,14-tetraazaheptadecan-1-oic acid Chemical compound NCCCC[C@H](C(=O)N[C@@H](C)C(O)=O)NC(=O)CC[C@H](C(N)=O)NC(=O)[C@@H](C)NC(=O)C(C)O[C@@H]1[C@@H](NCC)C(O)O[C@H](CO)[C@H]1O[C@H]1[C@H](NC(C)=O)[C@@H](O)[C@H](O)[C@@H](CO)O1 DQJCDTNMLBYVAY-ZXXIYAEKSA-N 0.000 description 1
- GOJUJUVQIVIZAV-UHFFFAOYSA-N 2-amino-4,6-dichloropyrimidine-5-carbaldehyde Chemical group NC1=NC(Cl)=C(C=O)C(Cl)=N1 GOJUJUVQIVIZAV-UHFFFAOYSA-N 0.000 description 1
- HRPVXLWXLXDGHG-UHFFFAOYSA-N Acrylamide Chemical compound NC(=O)C=C HRPVXLWXLXDGHG-UHFFFAOYSA-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
- 101100240657 Emericella nidulans (strain FGSC A4 / ATCC 38163 / CBS 112.46 / NRRL 194 / M139) swoF gene Proteins 0.000 description 1
- 241000620209 Escherichia coli DH5[alpha] Species 0.000 description 1
- 108090000368 Fibroblast growth factor 8 Proteins 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 101150094690 GAL1 gene Proteins 0.000 description 1
- 101150038242 GAL10 gene Proteins 0.000 description 1
- 102100028501 Galanin peptides Human genes 0.000 description 1
- 102100024637 Galectin-10 Human genes 0.000 description 1
- 108010015899 Glycopeptides Proteins 0.000 description 1
- 102000002068 Glycopeptides Human genes 0.000 description 1
- 102000003886 Glycoproteins Human genes 0.000 description 1
- 108090000288 Glycoproteins Proteins 0.000 description 1
- 102000051366 Glycosyltransferases Human genes 0.000 description 1
- 108700023372 Glycosyltransferases Proteins 0.000 description 1
- 101100121078 Homo sapiens GAL gene Proteins 0.000 description 1
- 101001046529 Homo sapiens Mevalonate kinase Proteins 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
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 1
- 101100536883 Legionella pneumophila subsp. pneumophila (strain Philadelphia 1 / ATCC 33152 / DSM 7513) thi5 gene Proteins 0.000 description 1
- CANPXOLVTMKURR-WEDXCCLWSA-N Lys-Gly-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H](N)CCCCN CANPXOLVTMKURR-WEDXCCLWSA-N 0.000 description 1
- 102000016943 Muramidase Human genes 0.000 description 1
- 108010014251 Muramidase Proteins 0.000 description 1
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 description 1
- 125000000729 N-terminal amino-acid group Chemical group 0.000 description 1
- 101100240662 Neurospora crassa (strain ATCC 24698 / 74-OR23-1A / CBS 708.71 / DSM 1257 / FGSC 987) gtt-1 gene Proteins 0.000 description 1
- 101150043338 Nmt1 gene Proteins 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 108010076504 Protein Sorting Signals Proteins 0.000 description 1
- 108020004511 Recombinant DNA Proteins 0.000 description 1
- 108020005091 Replication Origin Proteins 0.000 description 1
- 241000235347 Schizosaccharomyces pombe Species 0.000 description 1
- IXKSXJFAGXLQOQ-XISFHERQSA-N WHWLQLKPGQPMY Chemical compound C([C@@H](C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N1CCC[C@H]1C(=O)NCC(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(O)=O)NC(=O)[C@@H](N)CC=1C2=CC=CC=C2NC=1)C1=CNC=N1 IXKSXJFAGXLQOQ-XISFHERQSA-N 0.000 description 1
- PMZXXNPJQYDFJX-UHFFFAOYSA-N acetonitrile;2,2,2-trifluoroacetic acid Chemical compound CC#N.OC(=O)C(F)(F)F PMZXXNPJQYDFJX-UHFFFAOYSA-N 0.000 description 1
- 230000000145 adjuvantlike effect Effects 0.000 description 1
- 229960001230 asparagine Drugs 0.000 description 1
- 235000009582 asparagine Nutrition 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 239000013611 chromosomal DNA Substances 0.000 description 1
- 230000002759 chromosomal effect Effects 0.000 description 1
- 210000000349 chromosome Anatomy 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 210000004748 cultured cell Anatomy 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 230000003203 everyday effect Effects 0.000 description 1
- 230000001605 fetal effect Effects 0.000 description 1
- LXJXRIRHZLFYRP-UHFFFAOYSA-N glyceraldehyde 3-phosphate Chemical compound O=CC(O)COP(O)(O)=O LXJXRIRHZLFYRP-UHFFFAOYSA-N 0.000 description 1
- 230000006801 homologous recombination Effects 0.000 description 1
- 238000002744 homologous recombination Methods 0.000 description 1
- 230000001771 impaired effect Effects 0.000 description 1
- 230000005764 inhibitory process 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
- 210000003734 kidney Anatomy 0.000 description 1
- 229960000274 lysozyme Drugs 0.000 description 1
- 239000004325 lysozyme Substances 0.000 description 1
- 235000010335 lysozyme Nutrition 0.000 description 1
- 150000002704 mannoses Chemical class 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 238000000816 matrix-assisted laser desorption--ionisation Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000037353 metabolic pathway Effects 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 102000035118 modified proteins Human genes 0.000 description 1
- 108091005573 modified proteins Proteins 0.000 description 1
- 210000004897 n-terminal region Anatomy 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 230000002093 peripheral effect Effects 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 102000005162 pleiotrophin Human genes 0.000 description 1
- 230000009145 protein modification Effects 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 230000009962 secretion pathway Effects 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 238000012916 structural analysis Methods 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 230000005030 transcription termination Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 101150016309 trpC gene Proteins 0.000 description 1
- 239000002676 xenobiotic agent Substances 0.000 description 1
- 230000002034 xenobiotic effect Effects 0.000 description 1
- 210000005253 yeast cell Anatomy 0.000 description 1
Images
Classifications
-
- 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
- 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
-
- 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
-
- 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
- C12N5/10—Cells modified by introduction of foreign genetic material
-
- 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
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Biomedical Technology (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- Molecular Biology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Plant Pathology (AREA)
- Urology & Nephrology (AREA)
- Immunology (AREA)
- Medicinal Chemistry (AREA)
- Cell Biology (AREA)
- Hematology (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Gastroenterology & Hepatology (AREA)
- Toxicology (AREA)
- Food Science & Technology (AREA)
- Analytical Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Peptides Or Proteins (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Description
(1)下記の式1で表されるアミノ酸配列を含んでなること
X(−1)−X(0)・・・式1
ここでX(0)はThrまたはSerを表し、X(−1)はGly以外のアミノ酸を表す。
(2)タンパク質の高次構造解析において、スレオニンまたはセリンの側鎖がタンパク質表面に露出していると推定できること
(1)下記の式1で表されるアミノ酸配列を含んでなること
X(−1)−X(0)・・・式1
ここでX(0)はThrまたはSerを表し、X(−1)はGly以外のアミノ酸を表す
(2)タンパク質の高次構造解析において、セリンあるいはスレオニンの側鎖がタンパク質表面に露出していると推定できること
タンパク質をコードするcDNAは周知の方法で調整することができる。例えば、ヒトMKの場合には、例えば、Tsutsuiらの方法(Tsutsui, J. et al.: Biochem. Biophys. Res. Commun. 176, 792-797, 1991)により調製できる。また、適当なプライマーDNAを合成し、MKを発現している組織(例えば、腎臓、胎児脳)或いはヒト培養細胞(G401等)からRNAまたはcDNAを調製、これを鋳型としたPCR法により取得できる。他のタンパク質やペプチドを対象とする場合も同様に行える。
ヒト由来ミッドカイン(以下hMKと略す。)の酵母特異的O−グリコシレーションを受けるスレオニン周辺のアミノ酸を別なアミノ酸に置換し、ピキア・パストリス(P.pastoris)で発現させた場合の糖修飾の変化を解析することで、これらのアミノ酸配列が組換えhMKのO−グリコシレーションに影響を与えるかどうかを検討した。
5´側プライマー:
〔配列番号7〕
ccgaattcatgcagcaccgaggcttcctcc
3´側プライマー:
改変タイプ#1〔配列番号8〕
ccgaattctagtccttacccttacccttcttagccttagccttagccttagtttgtggagtacatggcttagtaacttggatggtctcctggcactgag
改変タイプ#2〔配列番号9〕
ccgaattctagtccttacccttacccttcttagccttagccttagccttagtttgtggagtacatggttgagtaacacggatagtttgctggcactgag
改変タイプ#3〔配列番号10〕
ccgaattctagtcctttcccttccctttcttggctttggcctttgctttggtaccgggggtgcagggcttggtaccgcggatggtttgctggcactgag
改変タイプ#4〔配列番号11〕
ccgaattctagtcctttcccttccctttcttggctttggcctttgctttggtaccgggggtgcagggcttggtaccgcgaccggtctcctggcactgag
改変タイプ#5〔配列番号12〕
ccgaattctagtcctttcccttccctttcttggctttggcctttgctttggtaccgggggtgcagggcttggtaccgcggatggtaccctggcactgag
実施例1においてスレオニンのN末側直近にグリシンが存在するとP.pastorisによる特異的な糖修飾が抑制される可能性が示唆された。しかしながら、この原因がアミノ酸の側鎖配列を認識していることによるのか、hMKタンパク質の高次構造の変化によるスレオニン側鎖の立体配座の変化であるためなのかは判断ができない。そこで、O−グリコシレーションの抑制がタンパク質の一次構造の違いにより生じたものなのかどうかを次に検討した。
〔配列番号13〕
hMK:Lys-Lys-Lys-Asp-Lys-Val-Lys-Lys
〔配列番号14〕
hMKTA:Lys-Thr-Lys-Thr-Lys-Thr-Lys-Lys
〔配列番号15〕
hMKGTA:Lys-Gly-Thr-Lys-Gly-Thr-Lys-Lys
〔配列番号16〕
ccgaattctagtcctttcccttccctttcttggctttggcctttgctttagccttgggggtgcagggcttagcgacgcggatagcctcctggc
hMKTA用5´側オリゴDNAプライマー
〔配列番号17〕
gaattcacaatgcaacaccgtggtttcctgttgctgaccttgctggctctgctggctttgacttccgctgtcgccaagactaagactaagactaagaagg
hMKGTA用5´側オリゴDNAプライマー
〔配列番号18〕
ggaattcacaatgcaacaccgtggtttcctgttgctgaccttgctggctctgctggctttgacttccgctgtcgccaagggtactaagggtactaagaag
タンパク質のO−グリコシレーションはタンパク質が翻訳され高次構造を形成した後に行われる。ゆえにO型糖修飾酵素がセリンあるいはスレオニンに糖を付加するには、修飾部位のアミノ酸側鎖の水酸基がタンパク質表面上に露出していることが必要条件であると思われた。この点を検討するためにはhMKの立体構造が既知である必要があるが、幸いなことにhMKタンパク質の立体構造はIwasaki等によってNMR解析され既知のものとなっており(Iwasaki, Nagata, et al., 1997)、これを参考にO−グリコシレーションされるスレオニン側鎖がどのような立体配座をとっているかを検討した。hMKはジスルフィド結合で形成された2つのドメイン構造(N末側のNドメインとC末側のCドメイン)とその両側のテイル部分からなる。hMKの立体構造はこれらNドメインとCドメイン、それぞれの分子構造をNMRで解析することにより決定された。Nドメインは3本、Cドメインは4本の逆平行βシート構造からなり、両ドメインの結合部とヘパリン結合部およびN末とC末のテイル部分は、明確な構造をとらないいわゆるループ構造(ランダムコイルともいう。)と考えられている。P.pastoris特異的に糖修飾を受けるCドメイン中のThr97、Thr101周辺の構造を見ると、どちらのスレオニン側鎖もhMKタンパク質表面上に非常に良く露出されていることがわかる(図6(a)(b))。これに対して同じCドメイン中にあって修飾されないThr76、Thr78、Thr84は、これらは偶然にも立体構造上では糖修飾されるThr97とThr101の直近に存在しており、その側鎖水酸基はタンパク質の内側を向くか他の側鎖に覆われるなどしてタンパク質表面に露出されていなかった。よって糖修飾されるスレオニン側鎖は、hMKタンパク質の表面に露出するという必要条件を満たしていたことになる。また、Thr108についてはC末テイル部分の高次構造が解析されていないためこの付近の立体構造の詳細は不明であるが、テイル部分はジスルフィド結合による制約を受けないループ構造と考えられているので、スレオニン側鎖が比較的自由に回転が出来るためにO−グリコシレーションされたと予想される。逆に近接するスレオニンが修飾されなかった理由は、スレオニン側鎖がタンパク質表面上に露出していないことが大きな原因であると予想される。
Claims (1)
- 酵母を用いてタンパク質あるいはペプチドを生産する際に起こる目的タンパク質あるいは目的ペプチドに対するO−グリコシレーションを回避する方法であって、以下の(1)及び(2)の要件を満たすX(−1)のアミノ酸をGlyに置換することを特徴とするO−グリコシレーションの回避方法。
(1)下記の式1で表されるアミノ酸配列を含んでなること
X(−1)−X(0)・・・式1
ここでX(0)はThrまたはSerを表し、X(−1)はGly以外のアミノ酸を表す。
(2)タンパク質の高次構造解析において、スレオニンまたはセリンの側鎖がタンパク質表面に露出していると推定できること
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2006510705A JP4772668B2 (ja) | 2004-03-03 | 2005-03-03 | 真核細胞におけるo−グリコシレーションの回避方法 |
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2004059898 | 2004-03-03 | ||
JP2004059898 | 2004-03-03 | ||
JP2006510705A JP4772668B2 (ja) | 2004-03-03 | 2005-03-03 | 真核細胞におけるo−グリコシレーションの回避方法 |
PCT/JP2005/003570 WO2005085433A1 (ja) | 2004-03-03 | 2005-03-03 | 真核細胞のo−グリコシレーションの制御方法 |
Publications (2)
Publication Number | Publication Date |
---|---|
JPWO2005085433A1 JPWO2005085433A1 (ja) | 2007-12-13 |
JP4772668B2 true JP4772668B2 (ja) | 2011-09-14 |
Family
ID=34917995
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP2006510705A Expired - Fee Related JP4772668B2 (ja) | 2004-03-03 | 2005-03-03 | 真核細胞におけるo−グリコシレーションの回避方法 |
Country Status (2)
Country | Link |
---|---|
JP (1) | JP4772668B2 (ja) |
WO (1) | WO2005085433A1 (ja) |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2021039574A1 (ja) * | 2019-08-23 | 2021-03-04 | 株式会社カネカ | O結合型糖鎖修飾が抑制された重鎖抗体 |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH0995454A (ja) * | 1995-10-02 | 1997-04-08 | Meiji Milk Prod Co Ltd | 抗潰瘍組成物 |
JP2002000276A (ja) * | 2000-06-28 | 2002-01-08 | Meiji Milk Prod Co Ltd | タンパク質のo−グリコシル化に関わるアミノ酸配列 |
-
2005
- 2005-03-03 JP JP2006510705A patent/JP4772668B2/ja not_active Expired - Fee Related
- 2005-03-03 WO PCT/JP2005/003570 patent/WO2005085433A1/ja active Application Filing
Also Published As
Publication number | Publication date |
---|---|
WO2005085433A1 (ja) | 2005-09-15 |
JPWO2005085433A1 (ja) | 2007-12-13 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
KR970005928B1 (ko) | 프라세싱 부위에 인접한 음전하를 띤 아미노산을 함유하는 이스트 프라세싱 시스템 | |
KR100227167B1 (ko) | 인체혈청알부민의 n-말단 단편을 함유하는 융합단백질 | |
JP5131666B2 (ja) | タンパク質の高分泌生産方法 | |
JP5737597B2 (ja) | 非天然コラーゲン様タンパク質及びその応用 | |
CA2127249A1 (en) | A human kunitz-type protease inhibitor variant | |
WO2001088143A1 (fr) | Procede d'elaboration de proteine avec reduction de chaine saccharose acide, et glycoproteine resultante | |
JP2001512684A (ja) | 新規のpichiapastoris遺伝子配列およびそれらの使用方法 | |
JP3366339B2 (ja) | 高効率発現ベクターを用いてサッカロミセスセレビジエから再組合蛋白質を製造する方法 | |
KR101291241B1 (ko) | 효모에서 인체 상피세포 성장인자를 대량 생산하는 방법 | |
JP4772668B2 (ja) | 真核細胞におけるo−グリコシレーションの回避方法 | |
EP0708112B1 (en) | RPDL protein and DNA encoding the same | |
PH27079A (en) | Immunogenic recombinant yeast expression product and method for purifying it | |
KR20030062854A (ko) | 분비형 벡터를 이용한 효모에서의 재조합 단백질의 제조방법 | |
JP5450985B2 (ja) | 真核細胞において組換えタンパク質を細胞外に放出させるためのポリヌクレオチド | |
JP2564764B2 (ja) | フィトラカインシュラリスナカイ由来の抗ウィルス性タンパク質遺伝子及びこれを発現する微生物 | |
JP3318602B2 (ja) | 糖鎖付加型ヘパリン結合性タンパク質、その製造方法およびそれを含有する医薬組成物 | |
Murasugi | Efficient expression and purification of recombinant therapeutic protein candidates, human midkine and pleiotrophin | |
Nam et al. | Expression and secretion of human lipocortin-1 by promoter and signal sequence of STA1 from Saccharomyces diastaticus | |
EP1230371B1 (de) | Verfahren zur herstellung von rekombinantem interleukin-8 und interleukin-8 muteinen und deren verwendung zum auffinden von antagonisten für il-8 rezeptoren | |
JP3136356B2 (ja) | 改変型シグナルペプチドをコードするdna | |
JP2002000276A (ja) | タンパク質のo−グリコシル化に関わるアミノ酸配列 | |
PT1666602E (pt) | Processo para a produção de uma proteína heteróloga utilizando células hospedeiras de uma espécie de levedura | |
US10655112B2 (en) | Polypeptide having endonuclease activity and method for producing the same | |
KR101623906B1 (ko) | 과립구 콜로니 자극인자 변이 단백질 또는 이의 트랜스페린 융합 단백질을 유효성분으로 포함하는 약학적 조성물 | |
WO2000009718A1 (fr) | Systeme de secretion/d'expression d'une grande quantite de proteines pures de la famille mk |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20080225 |
|
A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20101116 |
|
A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20110117 |
|
TRDD | Decision of grant or rejection written | ||
A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 Effective date: 20110621 |
|
A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 |
|
A61 | First payment of annual fees (during grant procedure) |
Free format text: JAPANESE INTERMEDIATE CODE: A61 Effective date: 20110622 |
|
FPAY | Renewal fee payment (event date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20140701 Year of fee payment: 3 |
|
R150 | Certificate of patent or registration of utility model |
Ref document number: 4772668 Country of ref document: JP Free format text: JAPANESE INTERMEDIATE CODE: R150 Free format text: JAPANESE INTERMEDIATE CODE: R150 |
|
S531 | Written request for registration of change of domicile |
Free format text: JAPANESE INTERMEDIATE CODE: R313531 |
|
R350 | Written notification of registration of transfer |
Free format text: JAPANESE INTERMEDIATE CODE: R350 |
|
LAPS | Cancellation because of no payment of annual fees |