KR100354344B1 - Hansenula polymorpha gene and strain lacking of the same - Google Patents
Hansenula polymorpha gene and strain lacking of the same Download PDFInfo
- Publication number
- KR100354344B1 KR100354344B1 KR1020020032933A KR20020032933A KR100354344B1 KR 100354344 B1 KR100354344 B1 KR 100354344B1 KR 1020020032933 A KR1020020032933 A KR 1020020032933A KR 20020032933 A KR20020032933 A KR 20020032933A KR 100354344 B1 KR100354344 B1 KR 100354344B1
- Authority
- KR
- South Korea
- Prior art keywords
- gene
- protease
- carboxypeptidase
- pep4
- polymorpha
- Prior art date
Links
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 101
- 241000320412 Ogataea angusta Species 0.000 title claims abstract description 22
- 101150029183 PEP4 gene Proteins 0.000 claims abstract description 54
- 102000005367 Carboxypeptidases Human genes 0.000 claims abstract description 44
- 108010006303 Carboxypeptidases Proteins 0.000 claims abstract description 44
- 101150107787 kex1 gene Proteins 0.000 claims abstract description 43
- 101710180012 Protease 7 Proteins 0.000 claims abstract description 40
- 101150050575 URA3 gene Proteins 0.000 claims abstract description 31
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 31
- 101100243377 Mus musculus Pepd gene Proteins 0.000 claims abstract description 24
- 239000013598 vector Substances 0.000 claims abstract description 11
- 241000336025 Ogataea parapolymorpha DL-1 Species 0.000 claims abstract description 9
- 239000013612 plasmid Substances 0.000 claims description 66
- 101500025419 Homo sapiens Epidermal growth factor Proteins 0.000 claims description 52
- 229940116978 human epidermal growth factor Drugs 0.000 claims description 47
- GVUGOAYIVIDWIO-UFWWTJHBSA-N nepidermin Chemical compound C([C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O)NC(=O)CNC(=O)[C@@H](NC(=O)[C@@H](NC(=O)[C@H](CS)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CS)NC(=O)[C@H](C)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](C)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CCSC)NC(=O)[C@H](CS)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CS)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CC(C)C)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CS)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC(N)=O)C(C)C)[C@@H](C)CC)C(C)C)C(C)C)C1=CC=C(O)C=C1 GVUGOAYIVIDWIO-UFWWTJHBSA-N 0.000 claims description 47
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 37
- 238000000034 method Methods 0.000 claims description 16
- 101100246753 Halobacterium salinarum (strain ATCC 700922 / JCM 11081 / NRC-1) pyrF gene Proteins 0.000 claims description 12
- 230000014509 gene expression Effects 0.000 claims description 11
- 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 4
- 230000028327 secretion Effects 0.000 claims description 3
- 101150028074 2 gene Proteins 0.000 claims description 2
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 2
- 238000012258 culturing Methods 0.000 claims 2
- 108010059081 Cathepsin A Proteins 0.000 abstract description 52
- 102000005572 Cathepsin A Human genes 0.000 abstract description 51
- 230000000694 effects Effects 0.000 abstract description 21
- 101150000922 PRC1 gene Proteins 0.000 abstract description 19
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 abstract description 13
- 230000015556 catabolic process Effects 0.000 abstract description 11
- 238000006731 degradation reaction Methods 0.000 abstract description 11
- 230000002950 deficient Effects 0.000 abstract description 7
- 108010076504 Protein Sorting Signals Proteins 0.000 abstract description 4
- 238000002360 preparation method Methods 0.000 abstract description 3
- 230000003248 secreting effect Effects 0.000 abstract description 2
- 102000002273 Polycomb Repressive Complex 1 Human genes 0.000 abstract 3
- 108010000598 Polycomb Repressive Complex 1 Proteins 0.000 abstract 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 43
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 description 42
- 210000004027 cell Anatomy 0.000 description 24
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 21
- 238000002105 Southern blotting Methods 0.000 description 21
- 150000001413 amino acids Chemical class 0.000 description 19
- 210000000349 chromosome Anatomy 0.000 description 17
- 235000018102 proteins Nutrition 0.000 description 17
- 108020004414 DNA Proteins 0.000 description 16
- 239000012634 fragment Substances 0.000 description 16
- 239000000523 sample Substances 0.000 description 16
- 235000001014 amino acid Nutrition 0.000 description 15
- 102000035195 Peptidases Human genes 0.000 description 14
- 108091005804 Peptidases Proteins 0.000 description 14
- 239000004365 Protease Substances 0.000 description 14
- 108091008146 restriction endonucleases Proteins 0.000 description 14
- 239000000243 solution Substances 0.000 description 13
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 12
- 238000004519 manufacturing process Methods 0.000 description 12
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 11
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 11
- 108010054576 Deoxyribonuclease EcoRI Proteins 0.000 description 9
- 239000002609 medium Substances 0.000 description 9
- 239000002773 nucleotide Substances 0.000 description 8
- 125000003729 nucleotide group Chemical group 0.000 description 8
- 238000004458 analytical method Methods 0.000 description 7
- 210000004899 c-terminal region Anatomy 0.000 description 7
- 238000010367 cloning Methods 0.000 description 7
- 238000010276 construction Methods 0.000 description 7
- 235000019419 proteases Nutrition 0.000 description 7
- 102000004190 Enzymes Human genes 0.000 description 6
- 108090000790 Enzymes Proteins 0.000 description 6
- 101150007280 LEU2 gene Proteins 0.000 description 6
- 229940088598 enzyme Drugs 0.000 description 6
- 101100494726 Neurospora crassa (strain ATCC 24698 / 74-OR23-1A / CBS 708.71 / DSM 1257 / FGSC 987) pep-4 gene Proteins 0.000 description 5
- 238000004128 high performance liquid chromatography Methods 0.000 description 5
- 239000002953 phosphate buffered saline Substances 0.000 description 5
- 108010025188 Alcohol oxidase Proteins 0.000 description 4
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 4
- ZHNUHDYFZUAESO-UHFFFAOYSA-N Formamide Chemical compound NC=O ZHNUHDYFZUAESO-UHFFFAOYSA-N 0.000 description 4
- 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 4
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 4
- FZWLAAWBMGSTSO-UHFFFAOYSA-N Thiazole Chemical compound C1=CSC=N1 FZWLAAWBMGSTSO-UHFFFAOYSA-N 0.000 description 4
- 239000011543 agarose gel Substances 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 238000011161 development Methods 0.000 description 4
- 239000008103 glucose Substances 0.000 description 4
- 238000002372 labelling Methods 0.000 description 4
- 239000000758 substrate Substances 0.000 description 4
- 230000009466 transformation Effects 0.000 description 4
- 230000014616 translation Effects 0.000 description 4
- 210000003934 vacuole Anatomy 0.000 description 4
- UAIUNKRWKOVEES-UHFFFAOYSA-N 3,3',5,5'-tetramethylbenzidine Chemical compound CC1=C(N)C(C)=CC(C=2C=C(C)C(N)=C(C)C=2)=C1 UAIUNKRWKOVEES-UHFFFAOYSA-N 0.000 description 3
- 108091026890 Coding region Proteins 0.000 description 3
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- 108010010803 Gelatin Proteins 0.000 description 3
- 241000235058 Komagataella pastoris Species 0.000 description 3
- 229910009891 LiAc Inorganic materials 0.000 description 3
- 108091028043 Nucleic acid sequence Proteins 0.000 description 3
- 239000001888 Peptone Substances 0.000 description 3
- 108010080698 Peptones Proteins 0.000 description 3
- 238000002835 absorbance Methods 0.000 description 3
- -1 arginine and lysine Chemical class 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- 229940041514 candida albicans extract Drugs 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- 239000003795 chemical substances by application Substances 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- 230000003247 decreasing effect Effects 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 239000008273 gelatin Substances 0.000 description 3
- 229920000159 gelatin Polymers 0.000 description 3
- 235000019322 gelatine Nutrition 0.000 description 3
- 235000011852 gelatine desserts Nutrition 0.000 description 3
- 238000012215 gene cloning Methods 0.000 description 3
- 101150066555 lacZ gene Proteins 0.000 description 3
- 244000005700 microbiome Species 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 235000019319 peptone Nutrition 0.000 description 3
- 238000012545 processing Methods 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 239000007787 solid Substances 0.000 description 3
- 230000001131 transforming effect Effects 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 239000012138 yeast extract Substances 0.000 description 3
- SEHFUALWMUWDKS-UHFFFAOYSA-N 5-fluoroorotic acid Chemical compound OC(=O)C=1NC(=O)NC(=O)C=1F SEHFUALWMUWDKS-UHFFFAOYSA-N 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 2
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 2
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 2
- 239000004475 Arginine Substances 0.000 description 2
- 241000222122 Candida albicans Species 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 2
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 2
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 2
- BACYUWVYYTXETD-UHFFFAOYSA-N N-Lauroylsarcosine Chemical compound CCCCCCCCCCCC(=O)N(C)CC(O)=O BACYUWVYYTXETD-UHFFFAOYSA-N 0.000 description 2
- 125000001429 N-terminal alpha-amino-acid group Chemical group 0.000 description 2
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 2
- 241000235648 Pichia Species 0.000 description 2
- 241000235070 Saccharomyces Species 0.000 description 2
- 241000235347 Schizosaccharomyces pombe Species 0.000 description 2
- 102000012479 Serine Proteases Human genes 0.000 description 2
- 108010022999 Serine Proteases Proteins 0.000 description 2
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- ISAKRJDGNUQOIC-UHFFFAOYSA-N Uracil Chemical compound O=C1C=CNC(=O)N1 ISAKRJDGNUQOIC-UHFFFAOYSA-N 0.000 description 2
- 206010052428 Wound Diseases 0.000 description 2
- 208000027418 Wounds and injury Diseases 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 239000008272 agar Substances 0.000 description 2
- 101150087698 alpha gene Proteins 0.000 description 2
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 2
- 239000002981 blocking agent Substances 0.000 description 2
- 229940095731 candida albicans Drugs 0.000 description 2
- 238000004132 cross linking Methods 0.000 description 2
- 239000012228 culture supernatant Substances 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000000855 fermentation Methods 0.000 description 2
- 230000004151 fermentation Effects 0.000 description 2
- PHTQWCKDNZKARW-UHFFFAOYSA-N isoamylol Chemical compound CC(C)CCO PHTQWCKDNZKARW-UHFFFAOYSA-N 0.000 description 2
- 230000005415 magnetization Effects 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 229920000728 polyester Polymers 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 230000002797 proteolythic effect Effects 0.000 description 2
- 229940024999 proteolytic enzymes for treatment of wounds and ulcers Drugs 0.000 description 2
- 238000011002 quantification Methods 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 125000003607 serino group Chemical group [H]N([H])[C@]([H])(C(=O)[*])C(O[H])([H])[H] 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 2
- 101150084750 1 gene Proteins 0.000 description 1
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- QRXMUCSWCMTJGU-UHFFFAOYSA-N 5-bromo-4-chloro-3-indolyl phosphate Chemical compound C1=C(Br)C(Cl)=C2C(OP(O)(=O)O)=CNC2=C1 QRXMUCSWCMTJGU-UHFFFAOYSA-N 0.000 description 1
- 101710188648 Acid trehalase Proteins 0.000 description 1
- 108030000961 Aminopeptidase Y Proteins 0.000 description 1
- 102000004580 Aspartic Acid Proteases Human genes 0.000 description 1
- 108010017640 Aspartic Acid Proteases Proteins 0.000 description 1
- 241000972773 Aulopiformes Species 0.000 description 1
- 125000001433 C-terminal amino-acid group Chemical group 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 108010076804 DNA Restriction Enzymes Proteins 0.000 description 1
- 238000007400 DNA extraction Methods 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 241000620209 Escherichia coli DH5[alpha] Species 0.000 description 1
- 108700039691 Genetic Promoter Regions Proteins 0.000 description 1
- HIZCTWCPHWUPFU-UHFFFAOYSA-N Glycerol tribenzoate Chemical compound C=1C=CC=CC=1C(=O)OCC(OC(=O)C=1C=CC=CC=1)COC(=O)C1=CC=CC=C1 HIZCTWCPHWUPFU-UHFFFAOYSA-N 0.000 description 1
- 102000007625 Hirudins Human genes 0.000 description 1
- 108010007267 Hirudins Proteins 0.000 description 1
- 108010017343 K2 killer toxin Proteins 0.000 description 1
- 101150045458 KEX2 gene Proteins 0.000 description 1
- 241001138401 Kluyveromyces lactis Species 0.000 description 1
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 1
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 1
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- 108010021466 Mutant Proteins Proteins 0.000 description 1
- 102000008300 Mutant Proteins Human genes 0.000 description 1
- 241000221961 Neurospora crassa Species 0.000 description 1
- 101150080255 PEP1 gene Proteins 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 102000003992 Peroxidases Human genes 0.000 description 1
- 229920001030 Polyethylene Glycol 4000 Polymers 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- 102000006382 Ribonucleases Human genes 0.000 description 1
- 108010083644 Ribonucleases Proteins 0.000 description 1
- 101900342676 Saccharomyces cerevisiae Carboxypeptidase Y Proteins 0.000 description 1
- 238000012300 Sequence Analysis Methods 0.000 description 1
- 208000007107 Stomach Ulcer Diseases 0.000 description 1
- 101000712605 Theromyzon tessulatum Theromin Proteins 0.000 description 1
- 229940122388 Thrombin inhibitor Drugs 0.000 description 1
- 239000007984 Tris EDTA buffer Substances 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
- 238000003277 amino acid sequence analysis Methods 0.000 description 1
- 210000004102 animal cell Anatomy 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- CKLJMWTZIZZHCS-REOHCLBHSA-N aspartic acid group Chemical group N[C@@H](CC(=O)O)C(=O)O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 239000006143 cell culture medium Substances 0.000 description 1
- 230000006037 cell lysis Effects 0.000 description 1
- 239000013611 chromosomal DNA Substances 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 239000007857 degradation product Substances 0.000 description 1
- 230000000593 degrading effect Effects 0.000 description 1
- 230000006866 deterioration Effects 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- UXGNZZKBCMGWAZ-UHFFFAOYSA-N dimethylformamide dmf Chemical compound CN(C)C=O.CN(C)C=O UXGNZZKBCMGWAZ-UHFFFAOYSA-N 0.000 description 1
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 229910052731 fluorine Inorganic materials 0.000 description 1
- 239000011737 fluorine Substances 0.000 description 1
- 230000027119 gastric acid secretion Effects 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 229940006607 hirudin Drugs 0.000 description 1
- WQPDUTSPKFMPDP-OUMQNGNKSA-N hirudin Chemical compound C([C@@H](C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC=1C=CC(OS(O)(=O)=O)=CC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CCCCN)NC(=O)[C@H]1N(CCC1)C(=O)[C@@H](NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)CNC(=O)[C@@H](NC(=O)[C@@H](NC(=O)[C@H]1NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CCC(O)=O)NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)CNC(=O)[C@H](CC(C)C)NC(=O)[C@H]([C@@H](C)CC)NC(=O)[C@@H]2CSSC[C@@H](C(=O)N[C@@H](CCC(O)=O)C(=O)NCC(=O)N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@H](C(=O)N[C@H](C(NCC(=O)N[C@@H](CCC(N)=O)C(=O)NCC(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCCCN)C(=O)N2)=O)CSSC1)C(C)C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H]1NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)CNC(=O)[C@H](CO)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CC=2C=CC(O)=CC=2)NC(=O)[C@@H](NC(=O)[C@@H](N)C(C)C)C(C)C)[C@@H](C)O)CSSC1)C(C)C)[C@@H](C)O)[C@@H](C)O)C1=CC=CC=C1 WQPDUTSPKFMPDP-OUMQNGNKSA-N 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 230000010354 integration Effects 0.000 description 1
- 230000037427 ion transport Effects 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- XIXADJRWDQXREU-UHFFFAOYSA-M lithium acetate Chemical compound [Li+].CC([O-])=O XIXADJRWDQXREU-UHFFFAOYSA-M 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 210000003712 lysosome Anatomy 0.000 description 1
- 230000001868 lysosomic effect Effects 0.000 description 1
- 230000013011 mating Effects 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 238000013365 molecular weight analysis method Methods 0.000 description 1
- CJERUMAUMMIPRF-FQEVSTJZSA-N n-[(2s)-3-(4-hydroxyphenyl)-1-(4-nitroanilino)-1-oxopropan-2-yl]benzamide Chemical compound C1=CC(O)=CC=C1C[C@@H](C(=O)NC=1C=CC(=CC=1)[N+]([O-])=O)NC(=O)C1=CC=CC=C1 CJERUMAUMMIPRF-FQEVSTJZSA-N 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 230000002018 overexpression Effects 0.000 description 1
- 108040007629 peroxidase activity proteins Proteins 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 239000003016 pheromone Substances 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 101150059999 pro gene Proteins 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 235000019833 protease Nutrition 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 239000012521 purified sample Substances 0.000 description 1
- 238000004451 qualitative analysis Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 238000004007 reversed phase HPLC Methods 0.000 description 1
- 235000019515 salmon Nutrition 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 229910000029 sodium carbonate Inorganic materials 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 239000003868 thrombin inhibitor Substances 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 238000002054 transplantation Methods 0.000 description 1
- 229940035893 uracil Drugs 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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)
- C12N9/58—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from fungi
- C12N9/60—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from fungi from yeast
-
- 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
- C07K14/475—Growth factors; Growth regulators
- C07K14/485—Epidermal growth factor [EGF], i.e. urogastrone
-
- 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
- C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
- C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
- C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Mycology (AREA)
- Wood Science & Technology (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Microbiology (AREA)
- Gastroenterology & Hepatology (AREA)
- Medicinal Chemistry (AREA)
- Plant Pathology (AREA)
- Physics & Mathematics (AREA)
- Toxicology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
본 발명은 한세눌라 폴리모르파 유전자 및 이들 유전자가 결핍된 균주의 제조에 있어서, 한세눌라 폴리모르파(Hansenula polymorpha)DL1 유래의 카르복시펩티다아제 Y를 코딩하는 유전자PRC1,프로티아제 A를 코딩하는 유전자PEP4유전자 및 카르복시펩티다아제 α를 코딩하는 유전자KEX1을 클로닝한 다음 이 유전자들중,PRC1과PEP4유전자를LEU유전자에 의해 디스럽션(disruption)시켜 제작한 벡터로 형질전환된 한세눌라 폴리모르파 UR2 카르복시펩티다아제 Y, 프로티아제 A 변이주를 제조하고 또PRC1, PEP4, KEX1유전자를 한세눌라 폴리모르파 URA3 유전자 팝아웃 카세트(pop-out cassette)에 의해 디스럽션시켜 제작한 벡터로 형질전환된 한세눌라 폴리모르파 DL1 카르복시펩티다아제 Y, 프로티아제 A, 카르복시펩티다아제 α변이주와 중복 변이주에 관한 것으로, 상기 제조한 균주들은 외래단백질 발현시 숙주세포로 사용하여 카르복시말단 분해가 감소된 외래 단백질을 제조하는 효과가 있고 또한 한세눌라 폴리모르파 유래의 분비 시그널 펩티드로서 사용할 수 있는 뛰어난 효과가 있다.The present invention provides a gene encoding the carboxypeptidase Y derived from Hansenula polymorpha DL1 , a gene encoding the PRC1, protease A, in the preparation of the Hansenula polymorpha gene and strains deficient in these genes. the cloned gene KEX1 encoding the PEP4 gene and carboxy peptidase α then transformed with a vector produced by illustration (disruption) am di by the genes of, PRC1 and PEP4 gene in LEU gene century Cronulla poly Maurepas UR2 carboxy Peptidase Y, Protease A Mutants were prepared and transformed with a vector produced by disrupting the PRC1, PEP4, KEX1 genes by Hansenula polymorpha URA3 gene pop-out cassette. Polymorph DL1 carboxypeptidase Y, protease A, carboxypeptidase α mutant and overlapping mutant, the preparation One strain has the effect of producing a foreign protein with reduced carboxy terminal degradation by using as a host cell when expressing a foreign protein, and also has an excellent effect of being used as a secretory signal peptide derived from Hanshenula polymorpha.
Description
본 발명은 한세눌라 폴리모르파 유전자 및 이들 유전자가 결핍된 균주에 관한 것이다. 더욱 상세하게는 한세눌라 폴리모르파(Hansenula polymorpha)DL1 유래의 카르복시펩티다아제 Y를 코딩하는 유전자PRC1, 프로티아제 A를 코딩하는PEP4유전자, 카르복시펩티다아제 α를 코딩하는KEX1유전자 및 이 유전자를 디스럽션(disruption)하여 제조한 카르복시 펩티다아제 Y, 프로티아제 A, 카르복시펩티다아제 α 결핍 균주에 관한 것이다.The present invention relates to a Hanshenula polymorpha gene and a strain deficient in these genes. More specifically century Cronulla poly Maurepas (Hansenula polymorpha) illustration am di the KEX1 gene, and a gene coding for the PEP4 gene, carboxy peptidase α encoding the gene PRC1, Pro thiazol agent A encoding the carboxy-peptidase Y of DL1-derived It relates to a carboxy peptidase Y, protease A, a carboxypeptidase α deficient strain prepared by (disruption).
재조합 단백질 생산 기술은 비교적 최근에 발달한 유전자 재조합 기술을 통하여 고등생물 유래의 유전자를 다양한 미생물로부터 대량 발현 생산하여 자연적으로 생산량이 한정된 고부가가치 의료용 단백질을 비교적 저렴한 비용으로 생산하는 기술로서 앞으로 난치성 질환의 증가와 국민 의료 수준의 제고에 따라 고 순도의 단백질성 의약품의 수요가 급증할 것으로 예상된다. 따라서 인체에 무해한 다양한 미생물을 이용하여 신 기능성 재조합 단백질을 비교적 저렴한 비용으로 대량 생산할 수 있는 기술개발 연구가 필요하다.Recombinant protein production technology is a technology that produces genes derived from higher organisms from various microorganisms in mass expression through relatively recently developed gene recombination technology to produce high value-added medical proteins with limited natural output at relatively low cost. The demand for high-purity proteinaceous drugs is expected to soar due to the increase and the improvement of national medical standards. Therefore, there is a need for a technology development research capable of mass production of a novel functional recombinant protein using a variety of microorganisms harmless to the human body at a relatively low cost.
효모를 숙주세포로 이용한 재조합 단백질생산 연구는 주로 전통효모 사카로마이세스 세레비지아에(Saccharomyces cerevisiae)를 이용하였으나 효율적인 외래단백질 발현을 위한 강력한 프로모터의 부재나 장시간 발효시에 야기되는 플라스미드의 불안정성 등의 이유로 재조합 단백질의 생산효율이 떨어지며 고농도 배양시 fed-batch 발효(fermentation)가 필요하며 발현된 이종 단백질들이 하이퍼글라이코실레이션(hyperglycosylation)이 된다는 점에서 비적절한 숙주로 간주되고 있다(Romanos, et al., Yeast, 8, 423, 1992). 이와 같은 단점을 보완하는 외래단백질 발현 시스템이 메탄올 자화 효모인 피치아 파스토리스(Pichia pastoris)에서 개발되었으며(Sudbery et al., Yeast, 10, 1707, 1994 ; Cregg et al., Bio/Technol. 5, 479, 1987) 최근에는 또다른 메탄올 자화 효모인 한세눌라 폴리모르파를 이용한 이종 단백질 발현 시스템의 개발 연구가 활발히 진행되고 있다(Gellissen et al., Bio/Technol. 9, 291, 1991; Janowicz et al., Yeast 7, 431, 1991). 새로운 대체숙주효모 중의 하나인 메탄올 자화 효모 한세눌라 폴리모르파는 값싼 원료물질인 메탄올을 탄소원으로 이용하여 비교적 쉽게 고농도 배양이 가능하며 메탄올 대사에 관련된 몇 가지 유전자 유래의 강력한 프로모터가 존재하고 또한 외래 유전자를 숙주세포의 염색체 DNA에 다중도입(multicopy integration)할 수 있어 고농도 배양동안에도 안정하게 유지되는 장점이 있다.Recombinant protein production research using yeast as a host cell mainly used the traditional yeast Saccharomyces cerevisiae, but the absence of a strong promoter for efficient foreign protein expression or plasmid instability caused by long-term fermentation. For this reason, the production efficiency of recombinant proteins is low, fed-batch fermentation is required in high concentration cultures, and heterologous proteins expressed are considered to be inadequate hosts because of their hyperglycosylation (Romanos, et. al., Yeast, 8, 423, 1992). An exogenous protein expression system was developed in Pichia pastoris , a methanol magnetizing yeast (Sudbery et al., Yeast, 10, 1707, 1994; Cregg et al., Bio / Technol. 5). , 479, 1987) Recently, the development of a heterologous protein expression system using another methanol magnetizing yeast Hansenula polymorpha has been actively studied (Gellissen et al., Bio / Technol. 9, 291, 1991; Janowicz et. al., Yeast 7, 431, 1991). Methanol magnetization yeast Hansenula polymorpha, one of the new alternative host yeasts, can be cultured relatively easily using methanol, a cheap raw material, as a carbon source, and has a strong promoter derived from several genes related to methanol metabolism. Multicopy integration into the chromosomal DNA of the host cell has the advantage of maintaining stable even during high concentration culture.
효모를 이용하여 재조합 단백질을 생산할 때 생산성의 증가를 위해서는 효율적인 발현 및 분비 시스템의 사용은 필수적이지만 생산 분비된 외래단백질을 단백질 분해효소 등으로부터의 분해를 방지하는 것도 매우 중요하다. 재조합 효모를 발효조에서 장시간 배양할 때 숙주세포로부터 자연적으로 분비되거나 또는 세포내에 존재하는 단백질 분해효소가 세포의 라이시스(lysis)를 통해서 배지로 나와 생산된재조합 단백질을 분해함으로써 전체적인 재조합 단백질의 생산성을 저하하는 문제가 있다. 재조합 단백질 생산 한세눌라 폴리모르파 세포 배양액에 분비된 재조합 단백질을 HPLC, MS 등을 통해 분석한 결과 상당부분의 재조합 단백질 카르복시 말단 분해산물이 나타났고 이는 숙주세포의 카르복시펩티다아제에 의해 재조합 단백질 카르복시말단의 아미노산이 1-2개 제거된 산물임이 관찰되었다.The use of efficient expression and secretion systems is essential to increase productivity when producing recombinant proteins using yeast, but it is also very important to prevent degradation of the produced and secreted foreign proteins from proteases and the like. When recombinant yeast is cultured in a fermenter for a long time, the protease secreted naturally from the host cell or present in the cell is released into the medium through cell lysis to degrade the produced recombinant protein, thereby improving overall productivity of the recombinant protein. There is a problem of deterioration. Recombinant protein production Recombinant protein secreted in Hanshenula polymorpha cell culture media was analyzed by HPLC, MS, etc., and a large amount of recombinant protein carboxyl terminal degradation products were detected. It was observed that the product was one to two amino acids removed.
사카로마이세스 세레비지아에의 액포(vacuole)는 고등세포의 리소좀(lysosome)에 해당하는 기관으로서 여러 가지 단백질 분해효소를 함유하고 있으며 영양분 고갈시 단백질분해 시스템을 담당하고 있다. 특히 카로복시펩티다아제 Y는 다양한 기질의 단백질을 분해할 수 있는 능력으로 인하여 카르복시말단 아미노산 분석에 이용되고 있으며 단백질의 분류나 위치를 연구하는 모델 단백질로서 많은 연구가 진행되어 있다(Rothman et al., Cell, 47, 1041, 1986 ; Johnson et al., Cell, 48, 875, 1987 ; Valls et al., J. Cell. Biol., 111, 361, 1992). 또한 외래 단백질의 과량 발현시 발생하는 카르복시말단의 분해는 카로복시펩티다아제 Y에 기인하는 것으로 알려져 있다. 카르복시펩티다아제 Y 유전자는 사카로마이세스 세레비지아에(Saccharomyces cerevisiae), 캔디다 알비칸스(Candida albicans), 피치아 파스토리스(Pichia pastoris), 쉬조사카로마이세스 폼베(Schizosaccharomyces pombe), 등에서 클로닝되어 공지되어 있다(Valls et al., Cell, 48, 887, 1987; Mukhtar et al., Gene, 121, 173, 1992; Ohi et al., Yeast, 12, 31, 1996; Tabuchi et al., J. Bacteriol., 179, 4179, 1997). 또 사카로마이세스 세레비지아에 프로티아제 A는 카로복시펩티다아제 Y와 함께 액포에존재하는 단백질 분해효소이다. 프로티아제 A는 액포에 존재하는 프로티아제 B, 카로복시펩티다아제 Y, 아미노펩티다아제 Y 같은 단백질 분해효소 뿐만 아니라 RNase, 알카라인 포스파타아제(alkaline phosphatase), 산 트레할라아제(acid trehalase)와 같은 액포 가수분해제(vacuolar hydrolase)의 단백질 가수분해 프로세싱(proteolytic processing)에 관여한다(H.B. Van Den Hazel et al.,Yeast.,12, 1, 1996). 특히PEP4유전자가 디스럽션 된 균주에서는 카르복시펩티다아제 Y의 활성도가 현저하게 감소하기 때문에 한세눌라 폴리모르파PEP4유전자를 클로닝하여 디스럽션 하면 카로복시펩티다아제 Y를 포함한 여러 분해효소의 활성을 저하시킬 수 있다.PEP4유전자는 사카로마이세스 세레비지아에(Saccharomyces cerevisiae), 캔디다 알비칸스(Candida albicans), 뉴로스포라 크라사(Neurospora crassa)등에서 클로닝되어 공지되어있다(Ammerer et al., Mol. Cell. Biol., 6, 2490, 1986; Woolford et al, Mol. Cell. Biol., 6, 25,1986; Lott et al., Nucleic Acids Res., 17, 1779, 1989; Bowman et al., Genbank accession No U36471).The vacuole of Saccharomyces cerevisiae is a lysosome of higher cells and contains various proteolytic enzymes and is responsible for the proteolytic system of nutrient depletion. In particular, carboxypeptidase Y is used for the analysis of carboxy terminal amino acids due to its ability to degrade proteins of various substrates, and many studies have been conducted as model proteins to study the classification and location of proteins (Rothman et al., Cell). , 47, 1041, 1986; Johnson et al., Cell, 48, 875, 1987; Valls et al., J. Cell. Biol., 111, 361, 1992). It is also known that degradation of the carboxy terminus resulting from overexpression of foreign proteins is due to carboxypeptidase Y. The carboxypeptidase Y gene has been cloned and known from Saccharomyces cerevisiae , Candida albicans , Pichia pastoris , Schizosaccharomyces pombe , and the like. (Valls et al., Cell, 48, 887, 1987; Mukhtar et al., Gene, 121, 173, 1992; Ohi et al., Yeast, 12, 31, 1996; Tabuchi et al., J. Bacteriol , 179, 4179, 1997). In addition, Saccharomyces cerevisiae protease A is a protease present in vacuoles with carboxypeptidase Y. Proteases A are proteases B, carboxypeptidase Y, aminopeptidase Y present in the vacuoles, as well as vacuoles such as RNase, alkaline phosphatase, and acid trehalase. It is involved in the proteolytic processing of a hydrolytic agent (HB Van Den Hazel et al., Yeast., 12, 1, 1996). In particular, since the activity of the carboxypeptidase Y is significantly reduced in the strains in which the PEP4 gene is disrupted, cloning and disruption of the Hanshenula polymorpha PEP4 gene may decrease the activity of various degrading enzymes including carboxypeptidase Y. have. The PEP4 gene is cloned and known from Saccharomyces cerevisiae , Candida albicans , Neurospora crassa , and others (Ammerer et al., Mol. Cell. Biol). ., 6, 2490, 1986; Woolford et al, Mol. Cell. Biol., 6, 25, 1986; Lott et al., Nucleic Acids Res., 17, 1779, 1989; Bowman et al., Genbank accession No U36471 ).
효모의KEX1유전자는 K1, K2 killer toxin 과 α-factor(mating pheromone) 전구체의 프로세싱(processing)에 관여하는 카르복시펩티다아제α를 코딩하는 것으로 알려져 있다(Alexandra et al., Cell, 50. 573, 1987). 카르복시펩티다아제α는 아르기닌(Arginine), 리신(Lysine)과 같은 염기성 아미노산으로 구성된 단백질의 카르복시말단을 특이적으로 분해하는 것으로 알려져 있으나 트롬빈 억제제인 히루딘(hirudin)을 사카로마이세스 세레비지아에에서 발현하였을 경우 카르복시말단이 염기성 아미노산인 경우뿐 아니라 티로신(Tyrosine), 류신(Leucine),글루타민(Glutamine)과 같은 비염기성 아미노산으로 구성된 경우에도 카르복시펩티다아제α에 의하여 카르복시말단이 분해되는 것이 밝혀졌다(Hinnen et al., Gene Expression in recombinant microorganism, 155-164, 1994). 따라서 한세눌라 폴리모르파에서도 사카로마이세스 세레비지아에의 경우와 동일한 기능을 수행 할 것으로 사료되어 한세눌라 폴리모르파 카르복시펩티다아제 α 유전자를 클로닝하여 카르복시펩티다아제 α 결핍 균주를 제조하고자 하였다.KEX1유전자는 사카로마이세스 세레비지아에(Saccharomyces cerevisiae), 피치아 파스토리스(Pichia pastoris), 쉬조사카로마이세스 폼베(Schizosaccharomyces pombe), 클루이베로마이세스 락티스(Kluyveromyces lactis)등에서 클로닝되어 공지되어있다(Bjourson et al., Cell, 50, 573, 1987., Boehm et al., Genbank accession No: AF095574., Lyne et al, Genbank accession No: AL023554., Tanguy-Rougeau et al., FEBS Lett, 234, 464, 1988)The yeast KEX1 gene is known to encode carboxypeptidase α, which is involved in the processing of K1, K2 killer toxin and α-factor (mating pheromone) precursors (Alexandra et al., Cell, 50. 573, 1987). . Carboxypeptidase α is known to specifically degrade the carboxy terminus of proteins consisting of basic amino acids, such as arginine and lysine, but the thrombin inhibitor hirudin in Saccharomyces cerevisiae When expressed, it was found that the carboxy terminal is decomposed by carboxypeptidase α not only when the carboxy terminal is a basic amino acid but also when it is composed of non-basic amino acids such as tyrosine, leucine, and glutamine. et al., Gene Expression in recombinant microorganism, 155-164, 1994). Therefore, Hanshenula polymorpha was expected to perform the same function as that of Saccharomyces cerevisiae, and to clone the Hanshenula polymorpha carboxypeptidase α gene to prepare a carboxypeptidase α deficient strain. KEX1 gene has been cloned and known from Saccharomyces cerevisiae , Pichia pastoris , Schizosaccharomyces pombe , Kluyveromyces lactis , etc. (Bjourson et al., Cell, 50, 573, 1987., Boehm et al., Genbank accession No: AF095574., Lyne et al, Genbank accession No: AL023554., Tanguy-Rougeau et al., FEBS Lett, 234, 464, 1988)
인체 표피 성장인자(human epidermal growth factor ; hEGF)는 53개의 아미노산으로 구성된 6 kDa의 단일 펩티드로 구성된 단백질로 표피 성장의 촉진 이외에 DNA와 단백질의 합성, 칼슘 전달에 의한 이온 수송, 위산 분비의 억제 등의 역할을 한다. 이러한 작용상의 특성에 의해 현재 창상이나 화상 치료, 각막 표층의 상처의 치료, 각막 이식 수술 후의 치료, 그리고 위궤양의 치료용으로 개발되고 있다. 따라서 이러한 약리학적 중요성과 동물 세포에서 극소량만이 추출되는데 착안하여 메탄올 자화 효모인 한세눌라 폴리모르파에서 대량으로 발현하여 분비시킨 후 카르복시 말단의 분해여부를 확인하였다. 종래에는 한세눌라 폴리모르파에서 인체 표피성장인자(hEGF)를 효과적으로 발현시키기 위하여, 먼저 hEGF 유전자를 포함하는 플라스미드 pUREGF를 제조하였다. pUREGF는 사카로마이세스 세레비지아에URA3유전자를 표지유전자로 사용하고 한세눌라 폴리모르파의 자기 복제 서열인 HARS36을 포함하고 있으며 메탄올 산화 효소(methanol oxidase)의 프로모터와 사카로마이세스 세레비지아에의 교배 인자 α 유래의 분비 신호인 프리프로 리더(pre-pro leader sequence), hEGF의 구조 유전자 및 메탄올 산화 효소의 터미네이터(terminator)가 순서대로 연결되어있는 플라스미드이다. pUREGF는 선택 표지유전자로 사카로마이세스 세레비지아에URA3유전자를 사용하기 때문에 hEGF 유전자의 다중도입을 유도할 수 있으며 메탄올 배지에서 가장 강력하게 유도되는 메탄올 산화 효소(methanol oxidase)의 프로모터를 사용하여 hEGF의 발현을 강력하게 유도할 수 있다. 또한 사카로마이세스 세레비지아에의 교배 인자 α 유래의 프리프로 리더뒤에 hEGF 유전자를 연결하여 발현된 hEGF 단백질을 세포 밖으로 분비시키는 특징이 있다. 한세눌라 폴리모르파 UR2 균주는 한세눌라 폴리모르파 DL1(leu,ura)균주를 플라스미드 pUREGF로 형질전환하여 hEGF 고 생산성 균주로 선별된 균주이다(대한민국 특허출원 제 97-54925호) .Human epidermal growth factor (hEGF) is a protein consisting of a single peptide of 6 kDa consisting of 53 amino acids. In addition to promoting epidermal growth, synthesis of DNA and proteins, ion transport by calcium transfer, inhibition of gastric acid secretion, etc. Plays a role. Due to these functional characteristics, they are currently being developed for the treatment of wounds and burns, the treatment of corneal superficial wounds, the treatment after corneal transplantation, and the treatment of gastric ulcers. Therefore, pharmacological significance and very small amount of animal cells were extracted, and the expression was secreted in large quantities in the methanol magnetization yeast Hansnurula polymorpha and confirmed the degradation of the carboxy terminal. Conventionally, in order to effectively express human epidermal growth factor (hEGF) in Hanshenula polymorpha, first, a plasmid pUREGF including the hEGF gene was prepared. pUREGF uses the URA3 gene as a marker gene in Saccharomyces cerevisiae and contains HARS36, a self-replicating sequence of Hanshenula polymorpha, a promoter of methanol oxidase and Saccharomyces cerevisiae. It is a plasmid in which a pre-pro leader sequence, a secretion signal derived from cross-linking factor α, a structural gene of hEGF, and a terminator of methanol oxidase are connected in sequence. pUREGF is due to the use of Saccharomyces URA3 gene in my process three Levy Jia as a selection marker gene can lead to multiple introduction of the hEGF gene, and using a promoter of methanol oxidase (methanol oxidase) it has been most highly induced in the methanol medium It can strongly induce the expression of hEGF. In addition, the hEGF protein is expressed outside the cell by linking the hEGF gene behind a prepro leader derived from a cross-linking factor α to Saccharomyces cerevisiae. The Hansenula polymorpha UR2 strain is a strain selected as a high productivity strain of hEGF by transforming Hansenula polymorpha DL1 (leu, ura) strain with plasmid pUREGF (Korean Patent Application No. 97-54925).
본 발명자들은 재조합 단백질 생산 한세눌라 폴리모르파에서 완전한 형태의 단백질을 생성하기 위해서 단백질 분해효소 결핍 변이균주를 개발하고자 하였다. 세포내에서 외래단백질의 카르복시 말단의 분해는 주로 카르복시펩티다아제 Y 및 카르복시펩티다아제 α에 의해서 행해지며 또한 카르복시펩티다아제 Y는 프로티아제 A에 의해서 진행(processing)되는 것으로 알려져 있으므로(Van Den Hazel etal., Yeast, 12, 1, 1996) 카르복시펩티다아제 Y, 카르복시펩티다아제 α와 프로티아제 A를 코딩하는 유전자인PRC1, KEX1과PEP4유전자를 각각 클로닝하였으며 클로닝된 이들 유전자를 이용하여 카르복시펩티다아제 Y, 카르복시펩티다아제 α, 프로티아제 A 결핍 변이주와 중복 변이주를 각각 제조하였다. 이들 변이주에서 외래단백질인 인체 표피 성장인자(Human epidermal growth factor : hEGF)를 발현하였을 때 카르복시말단이 분해된 단백질이 감소함을 확인함으로써 본 발명을 완성하게 되었다.The present inventors have attempted to develop protease deficient variant strains to produce a complete form of protein from recombinant protein-producing Hanshenula polymorpha. Degradation of the carboxy terminus of foreign proteins in cells is mainly carried out by carboxypeptidase Y and carboxypeptidase α and because carboxypeptidase Y is known to be processed by protease A (Van Den Hazel et al., Yeast , 12, 1, 1996) The carboxypeptidase Y, the carboxypeptidase α, and the PRC1, KEX1 and PEP4 genes encoding the proteases A were cloned, respectively, and the cloned genes were used for the carboxypeptidase Y, carboxypeptidase α, and protease. Thiase A deficient mutants and duplicate mutants were prepared, respectively. When the mutant proteins express human epidermal growth factor (hEGF) as foreign protein, the present invention was completed by confirming that the carboxy terminal decomposed protein decreased.
따라서, 본 발명의 목적은 한세눌라 폴리모르파 유래의 카르복시펩티다아제 Y, 카르복시펩티다아제 α 및 프로티아제 A를 코딩하는 유전자 염기서열을 동정하여 제공함에 있다. 본 발명의 다른 목적은 외래단백질 생산 한세눌라 폴리모르파 균주의 카르복시펩티다아제 Y를 코딩하는 유전자PRC1,카르복시펩티다아제 α를 코딩하는 유전자KEX1및 프로티아제 A를 코딩하는PEP4유전자가 각각 디스럽션된 벡터에 의해 형질전환된 카르복시펩티다아제 Y 변이주, 카르복시펩티다아제 α 변이주 및 프로티아제 A 변이주와 함께 이들 단백질분해효소가 중복하여 디스럽션된 균주를 제공함에 있다. 본 발명의 다른 목적은 상기 단백질분해효소 변이주를 숙주세포로 이용하여 카르복시 말단의 분해가 거의 발생하지 않은 외래 단백질 생산방법을 제공함에 있다.Accordingly, an object of the present invention is to identify and provide a gene sequence encoding carboxypeptidase Y, carboxypeptidase α, and protease A derived from Hanshenula polymorpha. Another object of the present invention is a vector in which the gene PRC1 encoding the carboxypeptidase Y, the gene KEX1 encoding the carboxypeptidase α, and the PEP4 gene encoding the protease A are disrupted, respectively, of the foreign protein producing Hanshenula polymorpha strain. A carboxypeptidase Y mutant, a carboxypeptidase α mutant, and a protease A mutant transformed by the present invention provide a strain in which these proteolytic enzymes overlapped. It is another object of the present invention to provide a foreign protein production method wherein the degradation of the carboxy terminus hardly occurs using the protease mutant strain as a host cell.
본 발명의 상기 목적은 사카로마이세스 세레비지아에 카르복시펩티다아제 Y유전자(PRC1)를 PCR을 이용하여 클로닝하고 이를 프로브로 이용하여 한세눌라 폴리모르파 DL-1의 염색체를 제한효소처리하여 서던블럿을 반복수행하므로써 한세눌라 폴리모르파PRC1유전자의 염기서열을 결정한 후 같은 방법으로 한세눌라 폴리모르파의 DL-1의 한세눌라 폴리모르파 DL-1의 프로티아제 A를 코딩하는 유전자(PEP4) 염기서열을 결정하고 한세눌라 폴리모르파KEX1유전자를 클로닝하기 위하여 균주간에 유사성이 높은 부위를 바탕으로 프라이머를 합성한 후 한세눌라 폴리모르파 염색체를 주형으로 PCR을 수행하여 한세눌라 폴리모르파KEX1유전자 단편을 클로닝 한 후 이를 프로브로 사용하여PRC1과 동일한 방법으로 한세눌라 폴리모르파KEX1전체 유전자를 클로닝한 다음 한세눌라 폴리모르파 UR2 균주의PRC1과PEP4의 유전자를 각각 디스럽션하기 위해 한세눌라 폴리모르파LEU2 유전자를 플라스미드 pHDY2, pHDP4에 각각 삽입하여 플라스미드 pHYL과 pHPL를 제조하고 이 플라스미드 pHYL과 pHPL로 한세눌라 폴리모르파 UR2 균주를 형질전환하여 카르복시펩티다아제 Y 변이주와 프로티아제 A 변이주를 얻고 이 변이주들의 카르복시펩티다아제의 활성을 측정한 후 서던블럿 분석으로 한세눌라 폴리모르파PRC1과PEP4유전자의 디스럽션을 확인하고 또 한세눌라 폴리모르파 DL1 균주의KEX1유전자를 디스럽션하여 하기 위해 한세눌라 폴리모르파URA3유전자 팝아웃 카세트을 플라스미드 pKH3.9에 삽입하여 플라스미드 pKUZ를 제조한 다음 한세눌라 폴리모르파 DL1 균주를 형질전환하여 카르복시펩티다아제 α 변이주를 얻고 한세눌라 폴리모르파 URA3 유전자가 팝아웃된 균주를 선별한 다음 야생타입 균주와 상기 선별한 변이균주의 염색체를 게놈서던 블럿하여KEX1유전자의 디스럽션을 확인하므로써 달성하였다. 또한 한세눌라 폴리모르파 DL1 균주의PRC1, PEP4유전자를 중복하여 디스럽션하기 위하여URA3유전자 팝아웃 카세트을 플라스미드 pHDY2와 pHDP4에 각각 삽입하여 플라스미드 pHTUZ와 pHPUZ를 각각 제조하고KEX1유전자 디스럽션과 같은 방법으로 형질전환과 팝아웃을 반복하여 프로티아제 A·카르복시펩티다아제 α변이주, 프로티아제 A·카르복시펩티다아제 Y 변이주, 카르복시펩티다아제 α·카르복시펩티다아제 Y 변이주, 프로티아제 A·카르복시펩티다아제 α·카르복시펩티다아제 Y 변이주를 각각 제조하였다.The object of the present invention is to clone a Saccharomyces cerevisiae carboxypeptidase Y gene ( PRC1 ) using PCR, and to use a probe as a probe to treat chromosomes of Hanshenula polymorpha DL-1. by the repeated century Cronulla poly Maurepas century Cronulla poly Maurepas pro gene thiazol encoding the agent a of DL-1 of the PRC1 DL-1 of a base and then determining the sequence century Cronulla poly know in the same way wave of genes (PEP4) In order to determine the nucleotide sequence and clone the Hanshenula polymorpha KEX1 gene, primers were synthesized based on high similarity between strains, and PCR was performed on the Hanshenula polymorpha chromosome as a template to perform the Hanshenula polymorpha KEX1 gene. After cloning the fragment, it was used as a probe to clone the entire gene of Hanshenula polymorpha KEX1 using the same method as PRC1. Next, in order to disrupt the genes of PRC1 and PEP4 of the Hanshenula polymorpha UR2 strain, the Hanshenula polymorpha LEU 2 gene was inserted into plasmids pHDY2 and pHDP4, respectively, to prepare plasmids pHYL and pHPL. a century Cronulla poly Maurepas by transforming the UR2 strain carboxy peptidase Y mutants and pro thiazol claim gained a mutant is carboxy century Cronulla polyester by Southern blot analysis and then measuring the activity of the peptidases of the mutant Maurepas PRC1 and the PEP4 gene di Plasmid pKUZ was prepared by inserting a Hanshenula polymorpha URA3 gene popout cassette into plasmid pKH3.9 to confirm the disruption and to disrupt the KEX1 gene of Hanshenula polymorpha DL1 strain. Transformation of the dl DL1 strain to obtain a carboxypeptidase α mutant strain and Hansenula Paul Maurepas URA3 gene was achieved by screening a strain and then pop out to the chromosome of the mutant and wild-type strains selected by the genomic Southern blot'm sure Sean D. KEX1 gene. Further century Cronulla poly know how each inserted into the URA3 gene pop-out kaseteueul plasmid pHDY2 and pHDP4 to redundantly PRC1, PEP4 gene of wave DL1 strain to design am di respectively produce plasmid pHTUZ and pHPUZ to and as KEX1 gene di am illustration Transformation and pop-out were repeated to protease A carboxypeptidase α mutant, protease A carboxypeptidase Y mutant, carboxypeptidase α carboxypeptidase Y mutant, protease A carboxypeptidase α carboxypeptidase Y Mutant strains were prepared respectively.
이하 본 발명의 구성 및 작용을 설명한다.Hereinafter, the configuration and operation of the present invention.
도 1은 한세눌라 폴리모르파PRC1유전자의 제한효소지도이다.1 is a restriction map of the Hanshenula polymorpha PRC1 gene.
도 2는 한세눌라 폴리모르파PEP4유전자의 제한효소지도이다.Figure 2 is a restriction map of the Hanshenula polymorpha PEP4 gene.
도 3은 한세눌라 폴리모르파KEX1유전자의 제한효소지도이다.3 is a restriction map of the Hanshenula polymorpha KEX1 gene.
도 4는 한세눌라 폴리모르파PRC1유전자를LEU2유전자를 이용하여 디스럽션하기 위하여 제작한 플라스미드의 제한효소 지도와 제작과정이다.Figure 4 is a restriction map and production process of the plasmid prepared for the disruption of Hanshenula polymorpha PRC1 gene using the LEU2 gene.
도 5는 한세눌라 폴리모르파PRC1유전자를URA3유전자를 이용하여 디스럽션하기 위하여 제작한 플라스미드의 제한효소 지도와 제작과정이다.FIG. 5 is a restriction map of the plasmid prepared for the disruption of the Hanshenula polymorpha PRC1 gene using the URA3 gene, and a manufacturing process.
도 6은 한세눌라 폴리모르파PEP4유전자를LEU2유전자를 이용하여 디스럽션하기 위하여 제작한 플라스미드의 제한효소 지도와 제작과정이다.Figure 6 is a restriction map of the plasmid and the production process in order to manufacture a century illustration am di Cronulla poly Maurepas PEP4 gene by the LEU2 gene.
도 7은 한세눌라 폴리모르파PEP4유전자를URA3유전자를 이용하여 디스럽션하기 위하여 제작한 플라스미드의 제한효소 지도와 제작과정이다.Figure 7 is a restriction map of the plasmid and the production process in order to manufacture a century illustration am di Cronulla poly Maurepas PEP4 gene by the URA3 gene.
도 8은 한세눌라 폴리모르파KEX1유전자를 디스럽션하기 위하여 제작한 플라스미드의 제한효소 지도와 제작과정이다.8 is a restriction map of the plasmid prepared for the disruption of Hanshenula polymorpha KEX1 gene and its production process.
도 9는 디스럽션된 유전자을 확인하기 위한 서던블럿 결과를 나타낸 사진도이다.9 is a photograph showing Southern blot results for identifying a disrupted gene.
본 발명은 공지의 사카로마이세스 세레비지아의 카로복시펩티다아제 Y를 코딩하는 유전자(PRC1)를 PCR을 이용하여 클로닝하고 이를 프로브로 이용하여 한세눌라 폴리모르파 DL-1의 염색체를 몇 가지 제한효소로 처리한 후 게놈 서던 하이브리디제이션(genomic Southern hybridization)함으로써 제한효소PstI으로 절단된 염색체의 3kb 위치에서PRC1유전자를 함유하는 DNA 밴드를 확인하였으며, 플라스미드에 삽입하여 라이브러리를 제조하고 서던블럿을 반복수행하여 한세눌라 폴리모르파PRC1유전자를 포함하는 플라스미드를 선별한 후 2.2kb의XhoI/PstI 단편으로 좁힌 플라스미드 pHDY2를 다시 선별한 다음PRC1유전자의 염기서열을 결정하는 단계; 공지의 사카로마이세스 세레비지아에PEP4유전자를 바탕으로 프라이머를 합성하고 PCR을 수행하여 사카로마이세스 세레비지아에PEP4유전자를 클로닝한 후 이를 프로브로 이용하여 한세눌라 폴리모르파 DL-1의 염색체를 몇 가지 제한효소로 처리한 다음 상기PRC1클로닝 방법과 동일한 방법으로 게놈 서던 하이브리디제이션(genomic Southern hybridization)을 실시하여 제한효소BamHI으로 절단된 염색체의 8kb 위치에서PEP4유전자를 함유하는 DNA 밴드를 확인하고 플라스미드에 삽입하여 라이브러리를 제조한 후 서던블럿을 반복 수행하여 한세눌라 폴리모르파PEP4유전자를 클로닝하는 단계; 여러 균주에서 밝혀진 카르복시펩티다아제 α의 아미노산 서열을 분석하여 균주간에 유사성이 높은 부위를 선별하고 그 부위를 바탕으로 프라이머를 합성한 후 한세눌라 폴리모르파 DL-1의 염색체를 주형으로 PCR을 수행하여 306bp 크기의 한세눌라 폴리모르파KEX1유전자 단편을 클로닝한 후 이를 프로브로 이용하여 한세눌라 폴리모르파 DL-1의 염색체를 몇 가지 제한효소로 처리한 다음 게놈 서던 하이브리디제이션(genomic Southern hybridization)하고 제한효소HindIII으로 절단된 염색체의 4.5kb 위치에서KEX1유전자를 함유하는 DNA 밴드를 확인하였으며, 플라스미드에 삽입하여 라이브러리를 제조하고 서던블럿을 반복수행하여 한세눌라 폴리모르파KEX1유전자를 포함하는 플라스미드를 선별한 후 3.9kb의EcoRI/HindIII 단편으로 좁힌 플라스미드 pKH3.9를 다시 선별한 다음KEX1유전자의 염기서열을 결정하는 단계; 상기 클로닝된 한세눌라 폴리모르파 DL-1의 카로복시펩티다아제 Y(PRC1) 및 프로티아제 A(PEP4) 유전자를 이용하여 염색체상의 이들 유전자들을 디스럽션(disruption)하기 위해 한세눌라 폴리모르파LEU2 유전자를 플라스미드 pHDY2 및 pHDP4에 각각 삽입하여 pHYL 및 pHPL을 제조한 후 플라스미드 pHYL과 pHPL을 한세눌라 폴리모르파 UR2 균주(leu2, hEGF,PRC1, PEP4,KEX1)에 형질전환하여 카르복시펩티다아제 Y 변이주(hEGF,prc1::LEU2,PEP4, KEX1)와 프로티아제 A 변이주(hEGF,pep4::LEU2,PRC1, KEX1)를 각각 제조하고 상기 제조한 카르복시펩티다아제 Y 변이주와 프로티아제 A 변이주 각각에 대해서 카르복시펩티다아제 Y 활성을 조사하고 야생 타입 균주와 상기 변이균주의 염색체를 게놈 서던 블럿하여 각각의 유전자가LEU2 유전자에 의해 디스럽션되었음을 확인하는 단계:KEX1, PEP4및PRC1유전자를 중복 디스럽션하기 위하여 한세눌라 폴리모르파URA3유전자 팝아웃 카세트을 플라스미드 pKH3.9, pHDY2, pHDP4에 각각 삽입하여 플라스미드pKUZ, pHYUZ, pHPUZ를 각각 제조하고 플라스미드 pKUZ을 한세눌라 폴리모르파 DL1 균주(leu2, ura3, KEX1, PEP4, PRC1)에 형질전환하여 카로복시펩티다아제α 변이주(leu2, kex1::URA3, PEP4, PRC1)를 제조한 후URA3유전자를 팝아웃하여 제거하고 순차적으로 pHYUZ와 pHPUZ를 형질전환하여 카르복시펩티다아제Y, 프로티아제A 변이 균주를 제조한 후 야생균주와 변이균주의 염색체를 이용하여 게놈 서던 블럿팅하여KEX1, PEP4, PRC1유전자가URA3유전자에 의해 디스럽션되었음을 확인하는 단계 및; 카르복시펩티다아제 Y 와 프로티아제 A 변이주에서 hEGF를 발현시킨 후 배양액을 원심 분리하고 HPLC 분석하여 상기 변이주의 세포밖으로 분비된 hEGF의 카르복시말단의 분해여부를 확인하는 단계로 구성된다.The present invention provides a gene encoding the known carboxypeptidase Y of Saccharomyces cerevisiae (PRC1) Cloning using PCR, and using this as a probe, the chromosome of Hanshenula polymorpha DL-1 was treated with several restriction enzymes, followed by genomic Southern hybridization.PstAt the 3 kb position of the chromosome cleaved by IPRC1DNA bands containing the genes were identified, inserted into the plasmid to prepare a library, and repeated Southern blotting.PRC1After selecting the plasmid containing the gene was 2.2kbXhoI /PstReselect the plasmid pHDY2 narrowed to the I fragmentPRC1Determining the base sequence of the gene; Notice of Saccharomyces cerevisiaePEP4Based on the gene, primers were synthesized and PCR was performed on Saccharomyces cerevisiae.PEP4After cloning the gene, the chromosome of Hanshenula polymorpha DL-1 was treated with several restriction enzymes using the probe.PRC1Genomic Southern hybridization was performed in the same manner as the cloning method to limit the enzyme.BamAt the 8 kb position of the chromosome cleaved with HIPEP4Check the DNA band containing the gene, insert it into the plasmid to prepare the library, and repeat the Southern blotting to perform Hansenula polymorpha.PEP4Cloning the gene; Analyze the amino acid sequence of carboxypeptidase α found in various strains, select sites with high similarity between the strains, synthesize primers based on the sites, and perform PCR using the chromosome of Hanshenula polymorpha DL-1 as a template. Hansenula polymorpha of sizeKEX1After cloning the gene fragment, the chromosome of Hansenula polymorpha DL-1 was treated with several restriction enzymes, followed by genomic Southern hybridization and restriction enzymes.Hinat 4.5 kb of the chromosome digested with dIIIKEX1DNA bands containing the genes were identified, inserted into the plasmid to prepare a library, and repeated Southern blotting.KEX1Plasmid containing the gene was screened andEcoRI /HinReselect plasmid pKH3.9 narrowed with dIII fragmentKEX1Determining the base sequence of the gene; The cloned Hansenula polymorpha DL-1 of carboxypeptidase Y (PRC1) And protease A (PEP4Hansenula polymorpha to disrupt these genes on the chromosome using genesLEU2 genes were inserted into plasmids pHDY2 and pHDP4, respectively, to prepare pHYL and pHPL.PRC1, PEP4,KEX1) was used to transform the carboxypeptidase Y mutant (hEGF,prc1 :: LEU2,PEP4, KEX1) And protease A mutants (hEGF,pep4 :: LEU2,PRC1, KEX1) And the carboxypeptidase Y mutant and protease A mutant prepared above For each, the carboxypeptidase Y activity was examined, and the genome of the wild type strain and the chromosome of the mutant strain was Southern blot.LEUStep 2 to confirm disruption by the gene:KEX1, PEP4AndPRC1Hanshenula polymorpha to duplicate genesURA3A gene popout cassette was inserted into plasmids pKH3.9, pHDY2, and pHDP4, respectively, to prepare plasmids pKUZ, pHYUZ, and pHPUZ, respectively, and plasmid pKUZ was used as a sensula polymorpha DL1 strain (leu2, ura3, KEX1, PEP4, PRC1) Transformed into a carboxypeptidase α mutant (leu2, kex1 :: URA3, PEP4, PRC1)URA3Population was removed by popping out genes, and then, by transforming pHYUZ and pHPUZ, carboxypeptidase Y and protease A mutant strains were prepared, followed by genomic Southern blotting using chromosomes of wild and mutant strains.KEX1, PEP4, PRC1GeneURA3Identifying that it has been disrupted by the gene; After expressing hEGF in the carboxypeptidase Y and protease A mutants, the culture medium is centrifuged and HPLC analysis to confirm the degradation of the carboxy terminal of hEGF secreted out of the mutant strain.
본 발명에서 형질전환된 카르복시펩티다아제 Y, 프로티아제 A 변이주 및 카르복시펩티다아제 α 변이주는 생명공학연구소내 유전자은행에 기탁하였다.The carboxypeptidase Y, protease A mutant and carboxypeptidase α mutants transformed in the present invention were deposited in the Gene Bank in the Biotechnology Research Institute.
이하, 본 발명의 구체적인 방법을 실시예를 들어 상세히 설명하고자 하지만 본 발명의 권리범위는 이들 실시예에만 한정되는 것은 아니다.Hereinafter, the specific method of the present invention will be described in detail with reference to Examples, but the scope of the present invention is not limited only to these Examples.
실시예 1: 카르복시펩티다아제 Y를 코딩하는 유전자Example 1 Gene Coding Carboxypeptidase Y PRC1PRC1 , 프로티아제 A를 코딩하는Coding for protease A PEP4PEP4 유전자 및 카르복시펩티다아제 α를 코딩하는 유전자Gene and gene encoding carboxypeptidase α KEX1KEX1 의 분리Separation of
제 1단계: 한세눌라 폴리모르파Step 1: Hansenula Polymorpha PRC1PRC1 ,, PEP4,PEP4, KEX1KEX1 유전자 클로닝을 위한 프로브 제작Probe Construction for Gene Cloning
본 단계에서는 하기 프라이머를 사용하여 사카로마이세스 세레비지아PRC1유전자를 클로닝하였다.In this step, the Saccharomyces cerevisiae PRC1 gene was cloned using the following primers.
프라이머 C1 ; 5'-ATG AAA GCA TTC ACC AG-3'Primer C1; 5'-ATG AAA GCA TTC ACC AG-3 '
프라이머 C2 ; 5'-TTA TAA GGA GAA ACC AC-3'Primer C2; 5'-TTA TAA GGA GAA ACC AC-3 '
즉, 상기 합성한 프라이머와 사카로마이세스 세레비지아의 염색체를 주형으로 94℃ 30초, 72℃ 2분 조건으로 25사이클의 PCR을 수행하여 1.6kb의 사카로마이세스 세레비지아PRC1유전자를 분리하였다. DIG-라벨링 검출 킷트(labeling and detection kit, Boehringer Mannheim사 제품)를 사용하여 제품 매뉴얼에 따라 라벨링하여 프로브를 제작하였다. 또 하기 프라이머를 사용하여 사카로마이세스 세레비지아PEP4유전자를 클로닝하였다.That is, 25 cycles of PCR were carried out at 94 ° C. for 30 seconds and 72 ° C. for 2 minutes using the synthesized primers and the chromosome of Saccharomyces cerevisiae as a template to obtain 1.6 kb of Saccharomyces cerevisiae PRC1 gene. Separated. Probes were made by labeling according to the product manual using a DIG-labeling detection kit (labeled and detection kit, manufactured by Boehringer Mannheim). In addition, the Saccharomyces cerevisiae PEP4 gene was cloned using the following primers.
프라이머 P1 ; 5'-ATG TTC AGC TTG AAA GC-3'Primer P1; 5'-ATG TTC AGC TTG AAA GC-3 '
프라이머 P2 ; 5'-TCA AAT TGC TTT GGC C-3'Primer P2; 5'-TCA AAT TGC TTT GGC C-3 '
즉, 상기 합성한 프라이머와 사카로마이세스 세레비지아의 염색체를 주형으로 94℃ 30초, 55℃ 30초, 72℃ 2분 조건으로 25사이클의 PCR을 수행하여 1.22kb의 사카로마이세스 세레비지아에PEP4유전자를 분리하였다. 상기 한세눌라 폴리모르파PRC1유전자 클로닝과 동일한 방법으로 라벨링하여 프로브를 제작하였다. 또 하기 프라이머를 사용하여 한세눌라 폴리모르파KEX1유전자를 클로닝하였다.That is, 25 cycles of PCR were carried out using the synthesized primer and the chromosome of Saccharomyces cerevisiae at 94 ° C 30 seconds, 55 ° C 30 seconds, and 72 ° C for 2 minutes. Levisias isolated the PEP4 gene. The probe was prepared by labeling in the same manner as the Hanshenula polymorpha PRC1 gene cloning. In addition, the following primer was used to clone the Hansenula polymorpha KEX1 gene.
프라이머 K1 ;5'-TGG YTS AAC GGH CCW GGH TGY TCB TCB-3'Primer K1; 5'-TGG YTS AAC GGH CCW GGH TGY TCB TCB-3 '
프라이머 K2 ;5'-WGG RAT GTA YTG WCC RGC GTA VGA CTC DCC-3'Primer K2; 5'-WGG RAT GTA YTG WCC RGC GTA VGA CTC DCC-3 '
즉, 상기 합성한 프라이머와 한세눌라 폴리모르파 염색체를 주형으로로 94℃ 30초, 50℃ 30초, 72℃ 30초 조건으로 5 사이클, 94℃ 30초, 55℃ 30초, 72℃ 30초 조건으로 20 사이클의 PCR을 수행하여 306bP의 한세눌라 폴리모르파KEX1유전자단편을 분리하였다. 상기 한세눌라 폴리모르파PRC1유전자 클로닝과 동일한 방법으로 라벨링하여 프로브를 제작하였다.That is, 5 cycles, 94 ° C 30 seconds, 55 ° C 30 seconds, 72 ° C 30 seconds under conditions of 94 ° C. 30 seconds, 50 ° C. 30 seconds, 72 ° C. 30 seconds, using the synthesized primer and the Hansenula polymorpha chromosome as a template. Under the condition, 20 cycles of PCR were performed to isolate a 306bP Hansenula polymorpha KEX1 gene fragment. The probe was prepared by labeling in the same manner as the Hanshenula polymorpha PRC1 gene cloning.
제 2단계: 한세눌라 폴리모르파 DL1의 게놈 DNA 추출Second Step: Genomic DNA Extraction of Hansenula Polymorpha DL1
존스톤의 방법 (Yeast Genetics, molecular aspects, pp.107-123, IRL Press, 1988)으로 YEPD 배지 (펩톤 2%, 효모추출물 1%, 포도당 2%)에서 배양한 한세눌라 폴리모르파 DL1 으로부터 게놈 DNA를 추출하였다.Genomic DNA from Hanshenula polymorpha DL1 cultured in YEPD medium (2% peptone, 1% yeast extract, 2% glucose) by Johnston's method (Yeast Genetics, molecular aspects, pp.107-123, IRL Press, 1988). Was extracted.
제 3단계: 플라스미드 pHDY2의 제작Step 3: Construction of Plasmid pHDY2
상기 2단계에서 얻은 게놈 DNA를 제한효소BamHI,EcoRI,EcoRV,HindIII,PstI,SalI으로 각각 처리하고, 0.9% 아가로스 겔에서 전기영동한 다음 Nytran membrane(Schleicher Schuell사 제품)으로 옮겨, 제 1단계에서 제조한 사카로마이세스 세레비지아PRC1유전자를 이용하여 제조한 프로브와 서던블럿하였다. 서던블럿 조건은 하이브리드액 (5X SSC, 0.1% N-lauryl sarcosine, 0.02% SDS, 2%blocking agent, 30% formamide)을 사용하여 42℃에서 6시간 반응시키는 것으로, Boehringer Mannheim사의 제품 매뉴얼에 따라 처리하였다. 알칼라인 포스파타제가 결합된 항체를 투여한 다음, BCIP와 X-phosphate를 첨가하여 보라색으로 염색된 밴드를 관찰하였다.PstI로 처리한 3kb 크기의 DNA에서 양성반응을 나타냈다. 밴드가 나타난 위치의 DNA를 추출하여 플라스미드 pBluescriptKSII+에 연결시킨 후 대장균 DH5α에 형질전환하여 라이브러리를 제조한 후 이 라이브러리를 대상으로 서던블럿을 반복하여PRC1유전자를 포함하는 플라스미드를 선별하였고 2.2 kb크기의XhoI/PstI 단편으로 축소하여 플라스미드 pHDY2을 제조하였다.PRC1유전자의 제한효소 지도와 염기서열 결정방법은 도 1에 나타내었고PRC1유전자의 염기서열을 결정한 결과는 서열 1에 나타내었다. DNA 서열은 GenBank에 U67174(96. 8. 17)로 등록하였다. 염기서열을 분석한 결과 한세눌라 폴리모르파 DL1PRC1유전자는 1,626 bp 크기로 인트론은 없으며 사카로마이세스 세레비지아에PRC1유전자와 54%의 유사성이 있으며, 염기서열을 바탕으로 유추한 아미노산 서열은 사카로마이세스 세레비지아에 카로복시펩티다아제 Y와 50%의 유사성을 나타내었다. 또한 세린 프로테아제(serine protease)의 활성 부위(active site)인 263번째 세린(serine) 잔기 부위에서도 높은 유사성을 나타내었다. The genomic DNA obtained in step 2 was treated with restriction enzymes Bam HI, Eco RI, Eco RV, Hin dIII, Pst I and Sal I, respectively, and electrophoresed on 0.9% agarose gel, followed by Nytran membrane (Schleicher Schuell) It was transferred to the Southern blot with a probe prepared using the Saccharomyces cerevisiae PRC1 gene prepared in the first step. Southern blotting conditions are 6 hours of reaction at 42 ° C using a hybrid solution (5X SSC, 0.1% N-lauryl sarcosine, 0.02% SDS, 2% blocking agent, 30% formamide), and treated according to Boehringer Mannheim's product manual. It was. An alkaline phosphatase conjugated antibody was administered, followed by addition of BCIP and X-phosphate to observe the purple stained band. Pst I treated with 3 kb sized DNA was positive. DNA was extracted from the band and linked to the plasmid pBluescriptKSII + and transformed into E. coli DH5α to prepare a library. Repeated Southern blot was used to select the plasmid containing the PRC1 gene. 2.2 kb Xho Plasmid pHDY2 was prepared by reduction to I / Pst I fragment. The restriction map of the PRC1 gene and the nucleotide sequence determination method are shown in FIG. 1, and the result of determining the nucleotide sequence of the PRC1 gene is shown in SEQ ID NO: 1. DNA sequence was registered in GenBank as U67174 (96. 8. 17). As a result of analyzing the nucleotide sequence, the Hanshenula polymorpha DL1 PRC1 gene is 1,626 bp in size and has no intron, and has 54% similarity to the PRC1 gene in Saccharomyces cerevisiae. Saccharomyces cerevisiae showed a 50% similarity with carboxypeptidase Y. It also showed high similarity at the 263th serine residue site, which is an active site of the serine protease.
제 4단계: 플라스미드 pHDP4의 제작Step 4: Construction of Plasmid pHDP4
상기 2단계의 DNA를 제한효소BamHI,EcoRI,EcoRV,HindIII,PstI,SalI으로 각각 처리하고, 0.9% 아가로스 겔에서 전기영동한 다음 Nytranmembrane(Schleicher Schuell사 제품)으로 옮겨, 제 1 단계에서 제조한 사카로마이세스 세레비지아PEP4유전자를 이용하여 제조한 프로브를 사용하여 제 3단계와 동일한 방법으로 서던블럿 하였다.BamHI로 처리한 8kb 크기의 DNA에서 양성반응을 나타냈다. 밴드가 나타난 위치의 DNA를 추출하여 제 3단계와 동일한 방법으로 라이브러리를 제조한 후 이 라이브러리를 대상으로 서던블럿을 반복하여PEP4유전자를 포함하는 플라스미드 pHDP3를 선별하였고 2.0kb크기의SacI/HindIII 단편으로 축소하여 플라스미드 pHDP4를 제조하였다.PEP4유전자의 제한효소 지도와 염기서열 결정방법은 도 2에 나타내었고PEP4유전자의 염기서열을 결정한 결과는 서열 2에 나타내었다. DNA 서열은 GenBank에 U67173(96. 8. 17)으로 등록하였다. 염기서열을 분석한 결과 한세눌라 폴리모르파 DL1PEP4유전자는 1,242 bp크기로 인트론은 없으며 사카로마이세스 세레비지아에PEP1유전자와 52.4 %의 유사성이 있으며, 염기서열을 바탕으로 유추한 아미노산 서열은 사카로마이세스 세레비지아에 프로티아제 A와 59%의 유사성을 나타내었다. 또한 아스파틸 프로테아제(aspartyl protease)의 활성 부위(active site)로 추정되는 117번째 아스파르트산(aspartic acid) 잔기 부위에서도 높은 유사성을 나타내었다.The DNA of step 2 was treated with restriction enzymes Bam HI, Eco RI, Eco RV, Hin dIII, Pst I, Sal I, electrophoresed on 0.9% agarose gel and then transferred to Nytranmembrane (Schleicher Schuell). Southern blot was performed in the same manner as in the third step using a probe prepared using the Saccharomyces cerevisiae PEP4 gene prepared in the first step. Positive results were observed in 8 kb sized DNA treated with Bam HI. After extracting DNA at the position where the band appeared, the library was prepared in the same manner as in step 3, and the Southern blot was repeated to select the plasmid pHDP3 containing the PEP4 gene, and the 2.0 kb Sac I / Hin dIII was selected. The fragment was reduced to make plasmid pHDP4. A restriction map and the sequencing method of the PEP4 gene, were shown in Figure 2 results determined the base sequence of the PEP4 gene are shown in SEQ ID NO: 2. DNA sequence was registered in GenBank as U67173 (96. 8. 17). As a result of the analysis of the nucleotide sequence, the Hansenula polymorpha DL1 PEP4 gene is 1,242 bp in size and has no intron, and it has 52.4% similarity to the PEP1 gene in Saccharomyces cerevisiae. Saccharomyces cerevisiae showed 59% similarity with protease A. It also showed high similarity at the 117th aspartic acid residue site, which is assumed to be the active site of aspartyl protease.
제 5단계: 플라스미드 pKH3.9의 제작Step 5: Construction of Plasmid pKH3.9
상기 2단계의 DNA를 제한효소BamHI,EcoRI,EcoRV,HindIII,PstI,SalI으로 각각 처리하고, 0.9% 아가로스 겔에서 전기영동한 다음 Nytran membrane(Schleicher Schuell사 제품)으로 옮겨, 제 1 단계에서 한세눌라 폴리모르파KEX1유전자를 이용하여 제조한 프로브를 사용하여 하이브리드액 조성을 (5X SSC, 0.1% N-lauryl sarcosine, 0.02% SDS, 2% blocking agent, 50% formamide)로 변경한 후 제 3단계와 동일한 방법으로 서던블럿하였다.HindⅢ로 처리한 4.5kb 크기의 DNA에서 양성반응을 나타냈다. 밴드가 나타난 위치의 DNA를 추출하여 제 3단계와 동일한 방법으로 라이브러리를 제조한 후 이 라이브러리를 대상으로 서던블럿을 반복하여KEX1유전자를 포함하는 플라스미드 pKH4.5를 선별하였고 3.9 kb크기의EcoRI/HindIII 단편으로 축소하여 플라스미드 pKH3.9를 제조하였다.KEX1유전자의 제한효소 지도와 염기서열 결정방법은 도 3에 나타내었고KEX1 유전자의 염기서열을 결정한 결과는 서열 3에 나타내었다. DNA 서열은 GenBank에 AF090325(98. 9. 4)으로 등록하였다. 염기서열을 분석한 결과 한세눌라 폴리모르파 DL1KEX1유전자는 인트론이 없는 1,833 bp 크기로 구성되어 있으며, 염기서열을 바탕으로 유추한 아미노산 서열은 사카로마이세스 세레비지아에 카르복시펩티다아제 α와 20%의 낮은 유사성을 나타내었다. 그러나 세린 프로테아제(serine protease)의 활성부위(active site)인 176번째 세린(serine) 잔기 부위에서는 카르복시펩티다아제 α 뿐만 아니라 카르복시펩티다아제 Y와도 높은 유사성을 나타내었으며 Von Heijne방법(Von Heijne, J. Mol. Biol., 173, 243, 1984)에 따라 아미노산 서열을 분석한 결과 18개의 시그널펩티드(signal-peptide)를 포함하고 있었다.DNA restriction enzyme of step 2BamHI,EcoRI,EcoRV,HindIII,PstI,SalEach was treated with I, electrophoresed on a 0.9% agarose gel and then transferred to a Nytran membrane (Schleicher Schuell), in the first step Hansenula polymorphaKEX1Using a probe prepared using a gene, the hybrid solution composition was changed to (5X SSC, 0.1% N-lauryl sarcosine, 0.02% SDS, 2% blocking agent, 50% formamide) and then Southern blotting was performed in the same manner as in the third step. It was.HinIt was positive for 4.5 kb DNA treated with dIII. After extracting the DNA where the band appeared, prepare a library in the same way as in step 3, and then repeat the Southern blot for this library.KEX1The plasmid pKH4.5 containing the gene was selected and 3.9 kb in size.EcoRI /HinThe plasmid pKH3.9 was prepared by reduction to the dIII fragment.KEX1Restriction enzyme map and nucleotide sequence determination method of the gene is shown in Figure 3KEX1 The result of determining the nucleotide sequence of the gene is shown in SEQ ID NO: 3. DNA sequence was registered in GenBank as AF090325 (98. 9. 4). Hanshenula polymorpha DL1 as a result of sequence analysisKEX1The gene is composed of 1,833 bp without intron, and the amino acid sequence inferred based on the nucleotide sequence shows 20% similarity to the carboxypeptidase α in Saccharomyces cerevisiae. However, the 176th serine residue, the active site of the serine protease, showed high similarity not only with carboxypeptidase α but also with carboxypeptidase Y. , 173, 243, 1984) and analyzed for amino acid sequences, which contained 18 signal-peptides.
실시예 2: 디스럽션된 플라스미드로 변이주 제조Example 2: Variation Preparation with Disrupted Plasmids
제 1단계: 디스럽션된 플라스미드 pHYL의 제작Step 1: Construction of Disrupted Plasmid pHYL
클로닝된 한세눌라 폴리모르파 DL-1의 카르복시펩티다아제 Y의 유전자인PRC1을 이용하여 염색체상의PRC1유전자를 디스럽션하기 위하여 도 4 에서 보는바와 같이 한세눌라 폴리모르파LEU2유전자가PRC1유전자에 삽입된 플라스미드 pHYL을 제조하였다. 먼저 제한효소EcoRI과BamHI으로 절단하여 추출한 1.2kb크기의 한세눌라 폴리모르파LEU2유전자를 Klenow 효소로 처리하여 평활 말단이 되도록 한 후PRC1유전자의 제한효소SmaI위치에 삽입하여 플라스미드 pHYL을 제작하였다.In order to disrupt the PRC1 gene on the chromosome using PRC1, which is the gene of the carboxypeptidase Y of the cloned Hanshenula polymorpha DL-1, as shown in FIG. 4, the Hanshenula polymorph LAU2 gene was inserted into the PRC1 gene. Plasmid pHYL was prepared. First, the 1.2 kb Hansenula polymorpha LEU2 gene, which was cut and extracted with the restriction enzymes Eco RI and Bam HI, was treated with Klenow enzyme so as to be blunt-ended, and inserted into the restriction enzyme Sma I position of the PRC1 gene to prepare plasmid pHYL. It was.
제 2단계: 디스럽션된 플라스미드 pHPL의 제작Second Step: Construction of Disrupted Plasmid pHPL
한세눌라 폴리모르파 DL-1의 프로티아제 A의 유전자인PEP4를 디스럽션 하기 위하여 도 6에 나타낸 바와같이 한세눌라 폴리모르파LEU2유전자로PEP4유전자의 일부분을 치환한 플라스미드 pHPL을 제조하였다. 먼저PEP4유전자를 제한효소EcoRV로 처리하여 1.05kb의PEP4유전자 단편을 제거한 후 제한효소EcoRI과BamHI으로 절단하여 추출한 1.2kb크기의 한세눌라 폴리모르파LEU2유전자를 Klenow 효소로 처리하여 평활 말단이 되도록 한 후 교체하여 플라스미드 pHPL을 제작하였다.Hansenula polymorpha DL-1 gene of the protease APEP4Hanshenula polymorpha as shown in FIG.LEU2By genePEP4Plasmid pHPL was prepared in which part of the gene was substituted. firstPEP4Gene restriction enzymeEco1.05kb processed by RVPEP4Restriction enzyme after removing the gene fragmentEcoRIBam1.2 kb Hanshenula polymorpha extracted with HILEU2Treat the gene with Klenow enzyme to make the blunt end The plasmid pHPL was produced by replacement.
제 3단계: 디스럽션된 플라스미드 pKUZ, pHYUZ, pHPUZ의 제작Step 3: Construction of Disrupted Plasmid pKUZ, pHYUZ, pHPUZ
클로닝된 한세눌라 폴리모르파 DL-1의KEX1유전자를 디스럽션하기 위하여 도 5에 나타낸 바와 같이 한세눌라 폴리모르파URA3유전자를 선택유전자로 반복사용하기 위한 팝아웃 카세트(pop-out cassette)를 제조하였다. 먼저 플라스미드 pUC19로부터 회수한 211bp의BamHI/PvuIILacZ유전자 단편을 1,323bp 크기의 한세눌라 폴리모르파URA3유전자 양 끝에 같은 방향이 되도록 연결하여 팝아웃 플라스미드 pLacUR3를 제조하였다. 플라스미드 pKH3.9를SmaI과EcoRI으로 처리하여 프로모터와 코딩부위를 일부 포함하는 1,184bp 단편을 제거하고 플라스미드 pLacUR3를PvuII와EcoRI으로 처리하여 얻은 211bp크기의 LacZ 유전자 다이렉트 리피트(direct repeats)를 포함하는 1,735bp 크기의 한세눌라 폴리모르파URA3유전자로 교체하여 플라스미드 pKUZ을 제조하였고, 플라스미드 pHDY2를SmaI과EcoRI으로 처리하여 코딩부위를 포함하는 1,055bp 단편을 제거하고 플라스미드 pLacUR3를PvuII와EcoRI으로 처리하여 얻은 1,735bp 크기의 한세눌라 폴리모르파URA3유전자로 교체하여 플라스미드 pHYUZ을 제조하였고, 플라스미드 pHDP4를XhoI과EcoRV으로 처리하여 코딩부위를 포함하는 303bp 단편을 제거하고 플라스미드 pLacUR3를PvuII와SalI으로 처리하여 얻은 1,800bp 크기의 한세눌라 폴리모르파URA3유전자로 교체하여 플라스미드 pHPUZ을 제조하였다. 먼저 플라스미드, pKUZ을 한세눌라 폴리모르파 DL1 균주(leu2, ura3, KEX1, PEP4, PRC1)에 형질전환하여 카르복시펩티다아제α 변이주(leu2, kex1::URA3, PEP4, PRC1)를 제조한 후 5-플루오로오로트산(5-Fluoroorotic acid)이 0.1 % 포함된 최소 고체배지(0.7% 아미노산이 결핍된 효모 기질(YNB), 2% 포도당, 0.1 % 5-플루오로오로트산(5-Fluoroorotic acid), 우라실(Uracil) 50㎍/mL, 로이신(Leucine) 50㎍/mL, 2% 아가)에서 72시간 이상 배양하여 한세눌라 폴리모르파URA3유전자가 팝아웃된균주(leu2, ura3, kex1::LacZ, PEP4, PRC1)를 선별하였고 이 균주를 플라스미드 pHYUZ로 형질전환하여 카르복시펩티다아제α·카르복시펩티다아제Y 변이주(leu2,kex1:: LacZ, prc1::URA3, PEP4)를 제조하였다 이균주를 다시 5-플루오로오로트산이 0.1 % 포함된 최소 고체배지에서URA3유전자가 팝아웃된 균주(leu2, ura3, kex1:: LacZ, prc1::LacZ, PEP4)를 선별하였고 플라스미드 pHPUZ를 사용하여 위와 같은 방법으로 카르복시펩티다아제α·카르복시펩티다아제Y·프로티아제 A 변이주(leu2, ura3, kex1::LacZ, prc1::LacZ, pep4::lacZ)를 제조하였다. 또한 플라스미드 pHYUZ와 pHPUZ를 사용하여 카르복시펩티다아제Y·프로티아제 A 변이주(leu2, ura3, prc1::LacZ, pep4::lacZ, KEX1)를, 플라스미드 pKUZ와 pHPUZ를 사용하여 카르복시펩티다아제α·프로티아제 A 변이주(leu2, ura3, kex1:: LacZ, pep4::lacZ, PRC1)를 각각 제조하였다.In order to disrupt the KEX1 gene of the cloned Hanshenula polymorpha DL-1, a pop-out cassette for repeatedly using the Hanshenula polymorpha URA3 gene as a selection gene is shown in FIG. Prepared. First, a popout plasmid pLacUR3 was prepared by linking 211 bp Bam HI / Pvu II LacZ gene fragment recovered from plasmid pUC19 in the same direction at both ends of the 1,323 bp Hansenula polymorpha URA3 gene. 211 bp LacZ gene direct repeats obtained by treatment of plasmid pKH3.9 with Sma I and Eco RI to remove 1,184 bp fragment containing a part of the promoter and coding region and plasmid pLacUR3 with Pvu II and Eco RI. The plasmid pKUZ was prepared by replacing with a 1,735 bp Hansenula polymorpha URA3 gene containing plasmid, and plasmid pHDY2 was treated with Sma I and Eco RI to remove the 1,055 bp fragment containing the coding region and plasmid pLacUR3 to Pvu II. Plasmid pHYUZ was prepared by replacing with 1,735 bp Hanshenula polymorpha URA3 gene obtained by treatment with Eco RI and plasmid pHDP4 was treated with Xho I and Eco RV to remove the 303 bp fragment containing the coding region and plasmid pLacUR3. by replacing a century Cronulla poly Maurepas URA3 gene of 1,800bp size obtained by treatment with Pvu II and Sal I Plastic A mid pHPUZ was prepared. First, the plasmid and pKUZ were transformed into polymorpha DL1 strains ( leu2, ura3, KEX1, PEP4, PRC1 ) to prepare carboxypeptidase α mutants ( leu2, kex1 :: URA3, PEP4, PRC1 ), and then 5-fluorine. Minimal solid medium containing 0.1% 5-Fluoroorotic acid (yeast substrate (YNB) lacking 0.7% amino acid, 2% glucose, 0.1% 5-Fluoroorotic acid) Cultured for more than 72 hours in uracil 50µg / mL, leucine 50µg / mL, 2% agar), and strains of Hansenula polymorpha URA3 gene popped out ( leu2, ura3, kex1 :: LacZ) , PEP4, PRC1 ) were selected and the strain was transformed with plasmid pHYUZ to prepare a carboxypeptidase α carboxypeptidase Y mutant ( leu2, kex1 :: LacZ, prc1 :: URA3, PEP4). URA3 gene popped out in a minimal solid medium containing 0.1% urorot acid ( leu2, ura3, kex1 :: LacZ, prc1 :: LacZ, PEP4 ) was selected and the carboxypeptidase α-carboxypeptidase Y-protease A mutants ( leu2, ura3, kex1 :: LacZ, prc1 :: LacZ, pep4 :: lacZ ) were prepared using the plasmid pHPUZ. . Moreover, the carboxypeptidase Y-protease A mutants ( leu2, ura3, prc1 :: LacZ, pep4 :: lacZ, KEX1 ) using plasmids pHYUZ and pHPUZ, and the carboxypeptidase α-proteases using plasmids pKUZ and pHPUZ A mutant strains ( leu2, ura3, kex1 :: LacZ, pep4 :: lacZ, PRC1 ) were prepared, respectively.
제 4단계: 형질전환Step 4: Transform
한세눌라 폴리모르파의 형질전환은 리튬 초산법에 따라 수행하였다. 즉, 한세눌라 폴리모르파 hEGF 고생산 균주인 UR2(leu2) 균주를 YEPD 액체배지(펩톤 2%, 효모 추출물 1%, 포도당 2%)에서 OD600=0.5까지 배양하고 세포를 회수하여 LiTE 용액(0.1 M Tris-Cl, pH8.0, 10mM EDTA, 10mM LiAc, pH7.5)으로 세척하였다. 다시 1/100부피의 LiTE 용액에 현탁하여 컴페턴트 세포(competent cell)를 제조하였다. 100μL의 컴페턴트 세포(competent cell)에 0.5μg의 플라스미드, 운반 DNA로 작용하는 10μg의 연어 정자 DNA와 0.6mL의 PEG/LiAc 용액(40 % PEG 4000, 0.1 M Tris-Cl, pH8.0, 10mM EDTA, 10mM LiAc, pH7.5)을 첨가하고 30℃에서 30분간 방치하였다. 70μL의 DMSO 용액을 첨가하고 42℃에서 15분간 반응 후 얼음에서 급속 냉각시키고 세포를 회수하여 최소 고체 배지(0.67% 아미노산이 결핍된 효모 기질, 2% 포도당, 2% 아가)에서 37℃로 72시간 배양하였다. 플라스미드 pHYL은 제한 효소XhoI/PstI으로 처리하여 선형화하고 플라스미드 pHPL은 제한 효소HindⅢ/NcoI으로, pKUZ는XhoI으로, pHYUZ는XhoI/PstI으로 pHPUZ는SpeI/SnaBI으로 선형화하여 형질전환에 사용하였다.Transformation of Hansenula polymorpha was performed according to the lithium acetate method. In other words, UR2 (leu2) strain, a high production strain of Hansenula polymorpha hEGF, was cultured in a YEPD liquid medium (2% peptone, 1% yeast extract, 2% glucose) to OD600 = 0.5, and cells were recovered to recover the LiTE solution (0.1). M Tris-Cl, pH8.0, 10 mM EDTA, 10 mM LiAc, pH7.5). Suspension was again suspended in 1/100 volume of LiTE solution to prepare competent cells. 0.5 μg plasmid in 100 μL competent cells, 10 μg salmon sperm DNA serving as carrier DNA and 0.6 mL PEG / LiAc solution (40% PEG 4000, 0.1 M Tris-Cl, pH8.0, 10 mM EDTA, 10 mM LiAc, pH7.5) was added and left at 30 ° C. for 30 minutes. Add 70 μL of DMSO solution and react for 15 minutes at 42 ° C., then rapidly cool on ice and recover the cells for 72 hours at 37 ° C. in minimal solid medium (yeast substrate lacking 0.67% amino acid, 2% glucose, 2% agar). Incubated. Plasmid pHYL was linearized by treatment with restriction enzyme Xho I / Pst I, plasmid pHPL was restriction enzyme Hin d Ⅲ / Nco I, pKUZ was Xho I, pHYUZ was Xho I / Pst I and pHPUZ was Spe I / Sna BI. Linearized and used for transformation.
실시예 3: 카르복시펩티다아제 Y 변이주, 프로티아제 A 변이주의 카르복시펩티다아제 Y 활성도 측정 및 이 균주로부터 생성된 hEGF 분석Example 3: Determination of Carboxypeptidase Y Activity of Carboxypeptidase Y Variants, Protease A Variants and Analysis of hEGF Generated from the Strain
제 1단계: 카르복시펩티다아제 Y, 프로티아제 A 변이주의 카르복시펩티다아제 Y 활성도 측정Step 1: Determination of Carboxypeptidase Y Activity of Carboxypeptidase Y and Protease A Mutants
디메틸포름아미드(Dimethylformamide)에 2.5 mg/mL로 녹인 N-벤조일-L-티로신-p-니트로아닐리드(N-benzoyl-L- tyrosine-p-nitroanilide)와 0.1M Tris-HCl (pH 7.5)를 1:4로 섞은 용액을 96웰 마이크로타이터플레이트(96 well microtiterplate)에 0.2 mL씩 분주한 후 각각의 웰에 실시예 2에서 얻은 형질전환체들을 접종하여 37℃에서 16시간동안 방치하였다. 카르복시펩티다아제의 활성이 있는 세포의 용액은 노란색을 띠며 활성이 없는 세포의 용액은 무색에 가까우므로 450nm에서 흡광도를 측정하여PRC1유전자가 효과적으로 디스럽션된 균주를 선별하였다. 또한 프로티아제 A 변이주에서는 카르복시펩티다아제 Y의 프로세싱(processing)이 저해되기때문에 카르복시펩티다아제의 활성을 측정함으로써PEP4유전자가 디스럽션된 균주를 선별하였다. 표 1에 나타낸 바와 같이 카르복시펩티다아제 Y 변이주에서 카르복시펩티다아제의 활성은 거의 나타나지 않았고, 프로티아제 A 변이주에서는 카르복시펩티다아제의 활성이 60%이상 감소하였다. 이로써 본 발명자들이 클로닝한PRC1과PEP4유전자가 한세눌라 폴리모르파 카르복시펩티다아제 Y, 프로티아제 A를 코딩하는 유전자임을 확인하였으며PRC1과PEP4유전자가 디스럽션된 균주에서 카르복시펩티다아제의 활성이 현저히 감소함을 확인하였다.N- dimethyl-benzoyl dissolved in 2.5 mg / mL in dimethylformamide (Dimethylformamide) -L- tyrosine -p- nitro anilide (N -benzoyl-L- tyrosine- p -nitroanilide ) with a 0.1M Tris-HCl (pH 7.5) 1 The solution mixed with: 4 was dispensed by 0.2 mL into a 96 well microtiterplate, and the wells of the transformants obtained in Example 2 were inoculated and left at 37 ° C. for 16 hours. Since the solution of the cells having the activity of carboxypeptidase is yellow and the solution of the cells with no activity is almost colorless, the absorbance was measured at 450 nm to select the strains that effectively disrupted the PRC1 gene. In addition, since the processing of carboxypeptidase Y was inhibited in the protease A mutant strain, strains in which the PEP4 gene was disrupted were selected by measuring the activity of carboxypeptidase. As shown in Table 1, the activity of the carboxypeptidase was hardly observed in the carboxypeptidase Y mutant, and the activity of the carboxypeptidase was reduced by 60% or more in the protease A mutant. This confirmed that the PRC1 and PEP4 genes cloned by the present inventors were genes encoding Hanshenula polymorpha carboxypeptidase Y and protease A. The activity of carboxypeptidase was significantly reduced in strains in which PRC1 and PEP4 genes were disrupted . It was confirmed.
제 2단계: 서던블럿에 의한 디스럽션 확인Step 2: Check for Disruption by Southern Blot
한세눌라 폴리모르파LEU2유전자에 의한 한세눌라 폴리모르파PRC1과PEP4유전자의 디스럽션 및 한세눌라 폴리모르파URA3유전자에 의한 한세눌라 폴리모르파KEX1유전자의 디스럽션을 확인하기 위하여 서던블럿을 수행하였다. 실시예 2의 4단계에서 선발한 형질전환체 각각을 YEPD 배지에 접종하여 37℃에서 18 ~ 20시간 진탕배양한 다음, 원심분리하여 세포를 회수하고, 1.5mL 튜브에 옮겼다. 세포를 STES 용액 (0.5M NaCl, 0.01M EDTA, 1% SDS in 0.2M Tris-Cl, pH 7.6) 30㎕에 현탁하고, 지름 0.4mm의 유리알을 0.8 부피만큼 첨가한 다음, 5분간 교반하고, TE 완충액 (1mM EDTA in 10mM Tris-Cl, pH 8.0) 200μL과 페놀/ 클로로포름/ 이소아밀알콜 (25:24:1) 200μL을 첨가하여 2분간 교반한 다음, 12,000rpm에서 원심분리하였다. 상등액에 2.5배 부피의 에탄올을 첨가하여 게놈 유전자를 침전시키고, 건조하였다. 게놈 유전자 2 ~ 3μg을 증류수 50μL에 녹이고, PRC1디스럽션 균주의 게놈 유전자는 제한효소EcoRI으로 처리하고PEP4디스럽션 균주의 게놈 유전자는 제한효소EcoRV으로 처리하고KEX1디스럽션 균주의 게놈 유전자는XhoI으로 처리한 후 0.8% 아가로스 겔에서 전기영동 하였다. 도 4, 도 6, 도 8에 표시한PRC1,PEP4, KEX1유전자를 프로브로 사용하여 서던블럿하여 밴드를 도 9에서 확인하였다. 카르복시펩티다아제 Y 변이주의 게놈 DNA를 제한효소EcoRI 으로 처리한 경우 0.65kb 크기의PRC1유전자 단편에LEU2유전자가 삽입되어 1.85kb크기로 증가하였다. 프로티아제 A 변이주의 게놈 DNA를 제한효소EcoRV로 처리한 경우PEP4유전자의 1.1kb의EcoRV 단편을LEU2유전자로 교체하는 과정에서EcoRV 인식부위가 제거되었기 때문에 10kb크기의 위치에서 단일 밴드를 확인할 수 있었다. 카르복시펩티다아제 α 변이주의 게놈 DNA를XhoI으로 처리한 경우 3.5kb 크기의KEX1유전자 단편에URA3유전자가 삽입되어 4kb크기로 증가하였으며 팝아웃된 균주에서는URA3유전자가 제거됨에 따라 2.5kb로 작아짐을 확인할 수 있었다. 따라서PRC1, PEP4유전자가 한세눌라 폴리모르파LEU2유전자에 의하여 디스럽션되었고KEX1유전자가URA3유전자에 의하여 디스럽션되었음을 확인하였다.The Southern blot for a century Cronulla poly Maurepas century Cronulla polyester according to the LEU2 gene Maurepas PRC1 and to confirm century design'm di Cronulla poly Maurepas KEX1 genes by di'm Sean and Hanse Cronulla poly Maurepas URA3 genes PEP4 gene Was performed. Each of the transformants selected in step 4 of Example 2 was inoculated in YEPD medium, shaken and cultured at 37 ° C. for 18 to 20 hours, and then the cells were recovered by centrifugation and transferred to a 1.5 mL tube. The cells are suspended in 30 μl of STES solution (0.5M NaCl, 0.01M EDTA, 1% SDS in 0.2M Tris-Cl, pH 7.6), 0.8 volume glass beads of 0.4 mm diameter are added, followed by stirring for 5 minutes, 200 μL of TE buffer (1 mM EDTA in 10 mM Tris-Cl, pH 8.0) and 200 μL of phenol / chloroform / isoamyl alcohol (25: 24: 1) were added and stirred for 2 minutes, followed by centrifugation at 12,000 rpm. Genomic genes were precipitated and dried by adding 2.5-fold volume of ethanol to the supernatant. Dissolve 2 to 3 μg of the genomic gene in 50 μL of distilled water, treat the genomic gene of the PRC1 disruption strain with the restriction enzyme Eco RI, and treat the genomic gene of the PEP4 disruption strain with the restriction enzyme Eco RV and the genome of the KEX1 disruption strain. Genes were treated with Xho I and electrophoresed on 0.8% agarose gel. Bands were identified in FIG. 9 by Southern blotting using the PRC1 , PEP4, and KEX1 genes shown in FIGS. 4, 6, and 8 as probes. When genomic DNA of the carboxypeptidase Y mutant strain was treated with the restriction enzyme Eco RI, the LEU2 gene was inserted into the PRC1 gene fragment having a size of 0.65 kb and increased to 1.85 kb. If the professional treatment of genomic DNA of the thiazole A mutant with restriction enzyme Eco RV a single band at the position of 10kb in size because the Eco RV recognition site is removed in the process of replacing the 1.1kb Eco RV fragment of the PEP4 gene by gene LEU2 I could confirm it. When the genomic DNA of the carboxypeptidase α mutant strain was treated with Xho I, the URA3 gene was inserted into the KEX1 gene fragment of 3.5 kb in size and increased to 4 kb in size, and decreased to 2.5 kb as the URA3 gene was removed from the popped out strain. there was. Therefore, PRC1, PEP4 gene was Sean D.'m century by Cronulla poly Maurepas LEU2 gene was confirmed that Sean KEX1 gene by the URA3 gene di am.
제 3단계: 본 발명 변이균주에서 분비된 hEGF의 분석Third step: analysis of hEGF secreted from the present invention strain
YP-메탄올 배지(효모 추출물 1%, 펩톤 2 %, 메탄올 2 %)에서 발현 분비된 hEGF의 분석을 위하여 배양액을 원심 분리하여 상등액을 분리하였다. 분리된 배양 상등액은 셉-팩 카트리지(Sep-Pak Cartridge, C18, Waters, Millipore사)에 의한 부분 정제를 거쳐 HPLC로 분석하였다. 셉-팩 부분 정제는 각각 10mL씩의 물, 메탄올, 0.1% 트리플로로아세트산, 20% 아세토니트릴/0.1% 트리플로로아세트산으로 처리하여 활성화된 Sep-Pak 카트리지에 20% 아세토니트릴/0.1% 트리플로로아세트산이 되도록 조정된 배양 상등액을 통과시켜 단백질을 흡착시킨 후, 20% 아세토니트릴, 0.1% 트리플로로아세트산으로 카트리지를 세척하여 불순물을 제거하였다. 흡착된 hEGF는 1mL의 50% 아세토니트릴/0.1% 트리플로로아세트산으로 3회에 거쳐 추출하였다. hEGF를 포함하는 추출액은 동결 건조하여 농축하였다. 부분 정제된 시료는 역상 HPLC(Beckman Model 126 System)를 이용하여 분석하였고, 시료의 분리를 위한 칼럼(column)은 4.6 × 250mm, 5㎛-C4 칼럼(Vydac사)을 사용하였다. 이동상의 유속은 0.8mL/분이었으며, 35분동안 20% 아세토니트릴/0.1% 트리플로로아세트산에서 60% 아세토니트릴/0.1% 트리플로로아세트산으로 농도를 증가 시켜 분리하였다. 이때 시료의 분리도는 215nm에서 흡광도를 측정하였다. HPLC에서 분리된 시료는 hEGF를 포함하고있는 피크(peak)를 확인하기 위하여 각 피크(peak)의 분획을 채취한 후, ELISA를 수행하였다. 채취한 시료를 항원 코팅 완충액(0.1M sodium carbonate buffer pH 9.6)에 적절히 희석하여 희석액 100μL씩을 마이크로타이터 플레이트웰(Nunc-immunomodule)에 넣고 37℃에서 2시간 동안 반응 시켰다. 웰을 PBST(PBS : phosphate buffered saline + 0.1% 트윈 20)로 씻어낸 후 PBS에 0.05%로 녹인 젤라틴을 웰당 100㎕씩 넣어 37℃에서 1시간 동안 반응시켰다. 상기 웰을 PBST로 씻어내고 hEGF의 단일 클론 항체(monoclonal antibody : UBI #05-109)를 0.05% 젤라틴이 들어있는 PBS 용액에 1:10,000의 비율로 희석하여 웰당 100μL씩 넣어 37℃에서 2시간동안 반응시킨 후 다시 PBST로 씻어내고 여기에 서양 고추냉이 퍼옥시다제가 결합된 이차 항체(Horse radish peroxidase conjugated goat anti-mouse IgG : Bio-Rad사)를 0.05% 젤라틴이 들어있는 PBS 용액에 1:3,000의 비율로 희석하여 웰당 100μL씩 넣어 37℃에서 1시간동안 반응시킨 후 PBST로 씻어내고 발색시켰다. 발색은 Pierce사의 퍼옥시다제 반응 기질인 TMB 기질 키트(Substrate Kit)를 사용하였는데, 0.04% TMB(3,3',5,5'tetramethyl benzidine)와 시트르산 완충액에 0.02%로 희석한 과산화수소를 1:1로 섞어 웰당 100μL씩 넣은 다음 약 15분 후 2M 황산을 100μL씩 넣어 반응을 정지시켰다. 발색시킨 후의 정량은 96-웰 플레이트 오토리더(96-well plate autoreader : THERMO max, Molecular Devices, USA)를 사용하여 450nm에서 흡광도를 측정하였으며, 정량을 위한 표준 hEGF 시료(recombinant hEGF : UBI #01-107)는 0.25ng/웰에서 5ng/웰까지 각각 희석하여 반응 시켰다. 실험결과, 두 개의 분획이 발현된 hEGF를 포함하고 있었으므로 이들의 정성분석을 위하여 채취한 분획을 동결건조 하였다. 두 가지 형태의 hEGF에 대하여 기초과학 지원 연구소에 N-말단 아미노산 서열 분석과 분자량 분석을 의뢰하였다. 분석 결과 두 개의 피크가 hEGF의 활성이 있는 것으로 나타났고, 두 개의 피크 가운데 비교적 친수성을 나타내는 피크는 MALDI-Mass 분석 결과 분자량이 6,205였으므로 53개의 아미노산으로 이루어진 완전한 hEGF였으며, 보다 더 높은 아세토니트릴 농도에서 용출되는, 완전한 hEGF보다는 소수성을 띠는 피크의 hEGF는 분자량이 6,053으로 측정되어 52개의 아미노산으로 이루어진 것으로 확인되었다. 이 두 가지의 hEGF는 N-말단 아미노산 서열 분석 결과 모두 Asn-Ser-Asp-Ser-Glu-로 이루어져 있으므로 시그널 펩티드로부터KEX2에 의해 정확하게 분리되었다. 이로부터 52개의 아미노산으로 이루어진 hEGF는 C-말단의 아미노산 한 개가 유리되어 형성된 것임을 알 수 있었으며, 이는C-말단의 아미노산 서열이 Arg-Leu-Glu-Trp-Trp-이므로 53개 아미노산의 완전한 hEGF에서 아르기닌이 유리되어 52개 아미노산의 hEGF가 되면 더 강한 소수성을 나타냄을 알 수 있었다. 상기 분석에 따라 HPLC 피크에 대한 정보를 확인한 후 한세눌라 폴리모르파 UR2 균주와 단백질 분해효소 결핍균주에서 분비된 hEGF의 조성을 비교하면, 프로티아제 A 변이주에서 발현된 hEGF는 UR2에서 발현된 hEGF에 비교할 때 전체적인 생성량이 감소하였으며 카르복시말단의 분해정도도 큰 변화를 확인할 수 없었다. 반면에 카르복시펩티다아제 Y 변이주에서는 카르복시펩티다아제 Y 디스럽션 효과를 확인할 수 있었다. UR2 균주에서 분비된 hEGF중 카르복시말단이 분해되지 않은 완전한 형태의 53개의 아미노산으로 구성된 hEGF은 37%이나 카르복시펩티다아제 Y 변이주에서는 53개의 아미노산으로 구성된 hEGF의 비율이 57%로 증가하였다.The supernatant was isolated by centrifugation of the culture for analysis of hEGF secreted in YP-methanol medium (1% yeast extract, 2% peptone, 2% methanol). The isolated culture supernatant was analyzed by HPLC after partial purification by Sep-Pak Cartridge (C18, Waters, Millipore). Sep-Pak partial tablets were treated with 20 mL acetonitrile / 0.1% triple in activated Sep-Pak cartridges with 10 mL each of water, methanol, 0.1% trifluoroacetic acid, 20% acetonitrile / 0.1% trifluoroacetic acid. After passing the culture supernatant adjusted to roroacetic acid to adsorb the protein, the cartridge was washed with 20% acetonitrile and 0.1% trifluoroacetic acid to remove impurities. The adsorbed hEGF was extracted three times with 1 mL of 50% acetonitrile / 0.1% trifluoroacetic acid. The extract containing hEGF was freeze-dried and concentrated. Partially purified samples were analyzed using reversed phase HPLC (Beckman Model 126 System), and a column for separation of samples was used as a 4.6 × 250 mm, 5 μm-C4 column (Vydac). The flow rate of the mobile phase was 0.8 mL / min and the concentration was separated from 20% acetonitrile / 0.1% trifluoroacetic acid to 60% acetonitrile / 0.1% trifluoroacetic acid for 35 minutes. At this time, the sample was measured for absorbance at 215 nm. Samples separated from HPLC were sampled from each peak to identify peaks containing hEGF, and then subjected to ELISA. The sample was properly diluted in antigen-coated buffer (0.1M sodium carbonate buffer pH 9.6), and 100 μL of the diluted solution was placed in a microtiter plate well (Nunc-immunomodule) and reacted at 37 ° C. for 2 hours. The wells were washed with PBST (PBS: phosphate buffered saline + 0.1% Tween 20), and 100 μl of gelatin dissolved in PBS at 0.05% was reacted at 37 ° C. for 1 hour. The wells were washed with PBST and monoclonal antibody (monoclonal antibody: UBI # 05-109) of hEGF was diluted in a ratio of 1: 10,000 in PBS solution containing 0.05% gelatin at a rate of 1: 10,000 and put in 100 μL per well for 2 hours at 37 ° C After the reaction, the resultant was washed again with PBST, and a horseradish peroxidase-conjugated secondary antibody (Horse radish peroxidase conjugated goat anti-mouse IgG: Bio-Rad) was added to a PBS solution containing 0.05% gelatin at 1: 3,000. After diluting at a rate of 100 μL per well and reacting at 37 ° C. for 1 hour, the resultant was washed with PBST and developed. Color development was performed using Pierce's peroxidase reaction substrate TMB Substrate Kit, which was diluted with 0.04% TMB (3,3 ', 5,5'tetramethyl benzidine) and 0.02% hydrogen peroxide in citric acid buffer 1: The mixture was mixed with 1 and added to 100 μL per well, and after about 15 minutes, 100 μL of 2M sulfuric acid was added thereto to stop the reaction. Quantification after color development was measured by absorbance at 450 nm using a 96-well plate autoreader (THRMO max, Molecular Devices, USA), and the standard hEGF sample (recombinant hEGF: UBI # 01-) for quantification. 107) was diluted from 0.25ng / well to 5ng / well and reacted. As a result, two fractions contained expressed hEGF, so the fractions collected for their qualitative analysis were lyophilized. N-terminal amino acid sequence analysis and molecular weight analysis were submitted to the Basic Science Support Institute for two types of hEGF. As a result, two peaks showed activity of hEGF. Among the two peaks, the relatively hydrophilic peak was MALDI-Mass analysis with a molecular weight of 6,205, resulting in a complete hEGF consisting of 53 amino acids and at a higher acetonitrile concentration. The eluted peak of the hydrophobic peak of hEGF, rather than the complete hEGF, was determined to have a molecular weight of 6,053 and was confirmed to consist of 52 amino acids. Both hEGFs were precisely separated from signal peptides by KEX2 since N-terminal amino acid sequencing both consisted of Asn-Ser-Asp-Ser-Glu-. From this, it can be seen that the hEGF consisting of 52 amino acids was formed by freeing one C-terminal amino acid, which means that in the complete hEGF of 53 amino acids, the amino acid sequence of the C-terminal is Arg-Leu-Glu-Trp-Trp-. Arginine was released and became a 52 amino acid hEGF was found to show a stronger hydrophobicity. After confirming the information on the HPLC peak according to the analysis and comparing the composition of the hEGF secreted from the Hansenula polymorpha UR2 strain and the protease-deficient strain, hEGF expressed in the protease A mutant strain was expressed in hEGF expressed in UR2 In comparison, the overall production decreased and the degree of degradation of the carboxy terminus could not be confirmed. On the other hand, in the carboxypeptidase Y mutant, it was confirmed that the carboxypeptidase Y disruption effect. Among the hEGFs secreted from the UR2 strain, hEGF consisting of 53 amino acids in which the carboxy-terminus was not degraded was 37%, but the ratio of hEGF consisting of 53 amino acids in the carboxypeptidase Y mutant increased to 57%.
상기의 실시예를 통하여 설명한 바와 같이 카르복시펩티다아제 Y를 코딩하는 유전자PRC1과 프로티아제 A를 코딩하는 유전자PEP4및 카르복시펩티다아제 α를코딩하는 유전자KEX1를 디스럽션한 유전자로 형질전환된 카르복시펩티다아제 Y 변이주, 프로티아제 A 변이주 및 카르복시펩티다아제 α 변이주 중, 카르복시펩티다아제 Y 변이주는 야생균주에 비하여 외래단백질인 hEGF의 카르복시말단 분해 능력을 40 %이상 감소시키고 카르복시펩티다아제 α 유전자가 추가로 디스럽션된 균주에서는 카르복시말단 분해 효과를 더 향상시킬 수 있는 효과가 있으며 프로티아제 A 변이주는 hEGF의 카르복시말단 분해 저하능력은 없지만 다른 외래단백질의 경우 카르복시말단 분해 저하 효과를 기대할 수 있는 뛰어난 효과가 있다. 그 뿐만 아니라 본 발명 재조합 카르복시펩티다아제 Y, 프로티아제 A, 카르복시펩티다아제 α는 한세눌라 폴리모르파 유래의 분비 시그널 펩티드로서 사용 가능한 효과가 있으므로 의약산업상 매우 유용한 발명인 것이다.Carboxypeptidase Y mutants transformed with a gene that disrupted the gene PRC1 encoding the carboxypeptidase Y, the gene PEP4 encoding the protease A and the gene KEX1 encoding the carboxypeptidase α as described above. Among the strains of the protease A and carboxypeptidase α mutants, the carboxypeptidase Y mutant reduced the carboxy terminal cleavage ability of the foreign protein hEGF by more than 40% and further disrupted the carboxypeptidase α gene compared to the wild strain. There is an effect that can further enhance the carboxy terminal degradation effect, and the protease A mutant has no ability to lower the carboxy terminal degradation of hEGF, but other foreign proteins have an excellent effect that can be expected to reduce the carboxy terminal degradation. In addition, the recombinant carboxypeptidase Y, protease A, and carboxypeptidase α of the present invention can be used as a secretory signal peptide derived from Hanshenula polymorpha, and thus are very useful inventions in the pharmaceutical industry.
Claims (14)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR1020020032933A KR100354344B1 (en) | 2002-06-12 | 2002-06-12 | Hansenula polymorpha gene and strain lacking of the same |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR1020020032933A KR100354344B1 (en) | 2002-06-12 | 2002-06-12 | Hansenula polymorpha gene and strain lacking of the same |
Related Parent Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| KR1019990007177A Division KR100354913B1 (en) | 1999-03-04 | 1999-03-04 | Hansenula polymorpha gene and strain lacking of the same |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| KR20020063145A KR20020063145A (en) | 2002-08-01 |
| KR100354344B1 true KR100354344B1 (en) | 2002-09-27 |
Family
ID=27726793
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| KR1020020032933A KR100354344B1 (en) | 2002-06-12 | 2002-06-12 | Hansenula polymorpha gene and strain lacking of the same |
Country Status (1)
| Country | Link |
|---|---|
| KR (1) | KR100354344B1 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR101498012B1 (en) * | 2012-06-19 | 2015-03-11 | 한국생명공학연구원 | HpGAS1 gene deleted yeasts and methods of producing recombinant proteins using them |
-
2002
- 2002-06-12 KR KR1020020032933A patent/KR100354344B1/en not_active IP Right Cessation
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR101498012B1 (en) * | 2012-06-19 | 2015-03-11 | 한국생명공학연구원 | HpGAS1 gene deleted yeasts and methods of producing recombinant proteins using them |
Also Published As
| Publication number | Publication date |
|---|---|
| KR20020063145A (en) | 2002-08-01 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US8853381B2 (en) | Signal peptide for producing a polypeptide | |
| US6861237B2 (en) | Production of heterologous polypeptides in yeast | |
| EP1313848B1 (en) | Yeast transformant producing recombinant human parathyroid hormone and method for producing the hormone | |
| KR20040002806A (en) | Hansenula polymorpha yapsin deficient mutant strain and process for the preparation of recombinant proteins using the same | |
| US20060121565A1 (en) | Methods for increasing the production of a recombinant polypeptide from a host cell | |
| KR100354344B1 (en) | Hansenula polymorpha gene and strain lacking of the same | |
| KR100371269B1 (en) | Hansenula polymorpha gene and strain lacking of the same | |
| KR100354913B1 (en) | Hansenula polymorpha gene and strain lacking of the same | |
| US6838555B2 (en) | Hansenula polymorpha mutants and process for the preparation of recombinant proteins using the same | |
| EP1546302A1 (en) | Methods for producing mammalian trypsins | |
| KR101920094B1 (en) | Compositions and Methods of Producing Enterokinase in Yeast | |
| US20030108908A1 (en) | Hansenula polymorpha mutants and process for the preparation of recombinant proteins using the same | |
| GB2249096A (en) | Saccharomyces cerevisiae strains lacking carboxypeptidase yscY activity for expression of proteins at high yields | |
| US8703444B2 (en) | Pichia pastoris deficient in endogenous secreted protease | |
| EP2906695B1 (en) | A nucleotide sequence and a process thereof | |
| Treerattrakool et al. | Secretion of Pem-CMG, a Peptide in the CHH/MIH/GIH Family of Penaeus monodon, in Pichia pastoris Is Directed by Secretion Signal of the α-Mating Factor from Saccharomyces cerevisiae | |
| RO et al. | Production of active carboxypeptidase Y of Saccharomyces cerevisiae secreted from methylotrophic yeast Pichia pastoris | |
| KR100436552B1 (en) | A Method for Measuring Substrate Specificity of Protease | |
| Cheng et al. | Recombinant protein secretion in Pseudozyma flocculosa and Pseudozyma antarctica with a novel signal peptide | |
| KR20040004089A (en) | Hansenula polymorpha mutant strains with defect in outer chain biosynthesis and the production of recombinant glycoproteins using the same strains | |
| KR20070027280A (en) | A method for surface display of target proteins using cell surface proteins of the yeast yarrowia | |
| JP2004089127A (en) | Method for producing protein | |
| JPH06253858A (en) | Fused gene having rodentian apolipoprotein e, plasmid comprising the same inserted thereinto and use thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A107 | Divisional application of patent | ||
| A201 | Request for examination | ||
| E701 | Decision to grant or registration of patent right | ||
| GRNT | Written decision to grant | ||
| FPAY | Annual fee payment |
Payment date: 20070914 Year of fee payment: 6 |
|
| LAPS | Lapse due to unpaid annual fee |