JP2021073883A - High productivity fc-binding protein, and method for producing the same - Google Patents
High productivity fc-binding protein, and method for producing the same Download PDFInfo
- Publication number
- JP2021073883A JP2021073883A JP2019202298A JP2019202298A JP2021073883A JP 2021073883 A JP2021073883 A JP 2021073883A JP 2019202298 A JP2019202298 A JP 2019202298A JP 2019202298 A JP2019202298 A JP 2019202298A JP 2021073883 A JP2021073883 A JP 2021073883A
- Authority
- JP
- Japan
- Prior art keywords
- amino acid
- seq
- binding protein
- fcrn
- acid sequence
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 108091008324 binding proteins Proteins 0.000 title claims abstract description 186
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 30
- 102000014914 Carrier Proteins Human genes 0.000 title claims abstract 16
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 135
- 238000006467 substitution reaction Methods 0.000 claims description 105
- 239000013604 expression vector Substances 0.000 claims description 40
- 108090000623 proteins and genes Proteins 0.000 claims description 39
- 125000000539 amino acid group Chemical group 0.000 claims description 37
- 235000001014 amino acid Nutrition 0.000 claims description 36
- 102000004169 proteins and genes Human genes 0.000 claims description 35
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 claims description 24
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 claims description 24
- 239000004472 Lysine Substances 0.000 claims description 24
- 235000013922 glutamic acid Nutrition 0.000 claims description 24
- 239000004220 glutamic acid Substances 0.000 claims description 24
- 235000003704 aspartic acid Nutrition 0.000 claims description 22
- CKLJMWTZIZZHCS-REOHCLBHSA-N aspartic acid group Chemical group N[C@@H](CC(=O)O)C(=O)O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 claims description 22
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 claims description 22
- 125000000291 glutamic acid group Chemical group N[C@@H](CCC(O)=O)C(=O)* 0.000 claims description 22
- 235000018102 proteins Nutrition 0.000 claims description 21
- 241000588724 Escherichia coli Species 0.000 claims description 15
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 claims description 15
- 235000004400 serine Nutrition 0.000 claims description 15
- 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 10
- 238000007792 addition Methods 0.000 claims description 10
- 238000012217 deletion Methods 0.000 claims description 8
- 230000037430 deletion Effects 0.000 claims description 8
- 238000003780 insertion Methods 0.000 claims description 8
- 230000037431 insertion Effects 0.000 claims description 8
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 claims description 7
- 125000003607 serino group Chemical group [H]N([H])[C@]([H])(C(=O)[*])C(O[H])([H])[H] 0.000 claims description 6
- 239000004474 valine Substances 0.000 claims description 6
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 claims description 5
- 239000004473 Threonine Substances 0.000 claims description 5
- 238000012258 culturing Methods 0.000 claims description 5
- 125000000341 threoninyl group Chemical group [H]OC([H])(C([H])([H])[H])C([H])(N([H])[H])C(*)=O 0.000 claims description 5
- 230000001131 transforming effect Effects 0.000 claims description 4
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 claims description 3
- 235000009582 asparagine Nutrition 0.000 claims description 3
- 229960001230 asparagine Drugs 0.000 claims description 3
- 125000000613 asparagine group Chemical group N[C@@H](CC(N)=O)C(=O)* 0.000 claims description 3
- 125000002987 valine group Chemical group [H]N([H])C([H])(C(*)=O)C([H])(C([H])([H])[H])C([H])([H])[H] 0.000 claims description 3
- 150000001413 amino acids Chemical class 0.000 abstract description 9
- 108091006020 Fc-tagged proteins Proteins 0.000 abstract description 5
- 102000023732 binding proteins Human genes 0.000 description 170
- 230000000052 comparative effect Effects 0.000 description 49
- 238000011156 evaluation Methods 0.000 description 44
- 238000000034 method Methods 0.000 description 34
- 241001198387 Escherichia coli BL21(DE3) Species 0.000 description 30
- 238000003752 polymerase chain reaction Methods 0.000 description 26
- 239000002609 medium Substances 0.000 description 18
- 108010068617 neonatal Fc receptor Proteins 0.000 description 18
- 229940027941 immunoglobulin g Drugs 0.000 description 17
- 210000004027 cell Anatomy 0.000 description 15
- 238000010494 dissociation reaction Methods 0.000 description 15
- 230000005593 dissociations Effects 0.000 description 15
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 14
- 239000000047 product Substances 0.000 description 14
- 108091008146 restriction endonucleases Proteins 0.000 description 13
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 10
- 239000000243 solution Substances 0.000 description 10
- 229930182817 methionine Natural products 0.000 description 9
- 239000002773 nucleotide Substances 0.000 description 9
- 125000003729 nucleotide group Chemical group 0.000 description 9
- 239000013598 vector Substances 0.000 description 9
- 239000004471 Glycine Substances 0.000 description 8
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 8
- PXHVJJICTQNCMI-UHFFFAOYSA-N Nickel Chemical compound [Ni] PXHVJJICTQNCMI-UHFFFAOYSA-N 0.000 description 8
- 229920002704 polyhistidine Polymers 0.000 description 8
- 108091034117 Oligonucleotide Proteins 0.000 description 7
- 238000006243 chemical reaction Methods 0.000 description 7
- 229930027917 kanamycin Natural products 0.000 description 7
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 7
- 229960000318 kanamycin Drugs 0.000 description 7
- 229930182823 kanamycin A Natural products 0.000 description 7
- 108020004705 Codon Proteins 0.000 description 6
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 6
- 229940024606 amino acid Drugs 0.000 description 6
- 238000002360 preparation method Methods 0.000 description 6
- 108010038807 Oligopeptides Proteins 0.000 description 5
- 102000015636 Oligopeptides Human genes 0.000 description 5
- 108010076504 Protein Sorting Signals Proteins 0.000 description 5
- 238000004458 analytical method Methods 0.000 description 5
- 238000004925 denaturation Methods 0.000 description 5
- 239000000284 extract Substances 0.000 description 5
- 125000001909 leucine group Chemical group [H]N(*)C(C(*)=O)C([H])([H])C(C([H])([H])[H])C([H])([H])[H] 0.000 description 5
- 239000000523 sample Substances 0.000 description 5
- 239000011780 sodium chloride Substances 0.000 description 5
- 238000002965 ELISA Methods 0.000 description 4
- 235000004279 alanine Nutrition 0.000 description 4
- 239000000872 buffer Substances 0.000 description 4
- 210000004899 c-terminal region Anatomy 0.000 description 4
- 238000005119 centrifugation Methods 0.000 description 4
- 239000012228 culture supernatant Substances 0.000 description 4
- 239000003446 ligand Substances 0.000 description 4
- 230000035772 mutation Effects 0.000 description 4
- 229910052759 nickel Inorganic materials 0.000 description 4
- 238000012216 screening Methods 0.000 description 4
- 239000011534 wash buffer Substances 0.000 description 4
- 239000004475 Arginine Substances 0.000 description 3
- 230000004544 DNA amplification Effects 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 3
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 description 3
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 3
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 description 3
- 238000012300 Sequence Analysis Methods 0.000 description 3
- 238000002835 absorbance Methods 0.000 description 3
- 238000001042 affinity chromatography Methods 0.000 description 3
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 3
- 229940041514 candida albicans extract Drugs 0.000 description 3
- 238000004587 chromatography analysis Methods 0.000 description 3
- 235000018417 cysteine Nutrition 0.000 description 3
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 3
- 229960002433 cysteine Drugs 0.000 description 3
- 230000036425 denaturation Effects 0.000 description 3
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 3
- 125000000487 histidyl group Chemical group [H]N([H])C(C(=O)O*)C([H])([H])C1=C([H])N([H])C([H])=N1 0.000 description 3
- MGFYIUFZLHCRTH-UHFFFAOYSA-N nitrilotriacetic acid Chemical compound OC(=O)CN(CC(O)=O)CC(O)=O MGFYIUFZLHCRTH-UHFFFAOYSA-N 0.000 description 3
- 239000008363 phosphate buffer Substances 0.000 description 3
- 239000000758 substrate Substances 0.000 description 3
- 230000009466 transformation Effects 0.000 description 3
- 239000012138 yeast extract Substances 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 2
- 102100026120 IgG receptor FcRn large subunit p51 Human genes 0.000 description 2
- 108060003951 Immunoglobulin Proteins 0.000 description 2
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 2
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 description 2
- 239000006142 Luria-Bertani Agar Substances 0.000 description 2
- 108700018351 Major Histocompatibility Complex Proteins 0.000 description 2
- 229920001213 Polysorbate 20 Polymers 0.000 description 2
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 description 2
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 2
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 2
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 2
- 239000003463 adsorbent Substances 0.000 description 2
- 125000000637 arginyl group Chemical group N[C@@H](CCCNC(N)=N)C(=O)* 0.000 description 2
- 102000015736 beta 2-Microglobulin Human genes 0.000 description 2
- 108010081355 beta 2-Microglobulin Proteins 0.000 description 2
- 239000013522 chelant Substances 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 230000001419 dependent effect Effects 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 238000001641 gel filtration chromatography Methods 0.000 description 2
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 description 2
- 125000003630 glycyl group Chemical group [H]N([H])C([H])([H])C(*)=O 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 102000018358 immunoglobulin Human genes 0.000 description 2
- 239000000411 inducer Substances 0.000 description 2
- 238000010255 intramuscular injection Methods 0.000 description 2
- 239000007927 intramuscular injection Substances 0.000 description 2
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 description 2
- 229960000310 isoleucine Drugs 0.000 description 2
- 238000004811 liquid chromatography Methods 0.000 description 2
- 125000003588 lysine group Chemical group [H]N([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 235000015097 nutrients Nutrition 0.000 description 2
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 2
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 2
- 235000013930 proline Nutrition 0.000 description 2
- 238000000751 protein extraction Methods 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 230000028327 secretion Effects 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 235000020183 skimmed milk Nutrition 0.000 description 2
- 230000020382 suppression by virus of host antigen processing and presentation of peptide antigen via MHC class I Effects 0.000 description 2
- 238000002198 surface plasmon resonance spectroscopy Methods 0.000 description 2
- 239000012137 tryptone Substances 0.000 description 2
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 description 1
- 238000009020 BCA Protein Assay Kit Methods 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 108700010070 Codon Usage Proteins 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 241000672609 Escherichia coli BL21 Species 0.000 description 1
- 241000701959 Escherichia virus Lambda Species 0.000 description 1
- 108010024636 Glutathione Proteins 0.000 description 1
- 102000005720 Glutathione transferase Human genes 0.000 description 1
- 108010070675 Glutathione transferase Proteins 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 101000913079 Homo sapiens IgG receptor FcRn large subunit p51 Proteins 0.000 description 1
- 101710177940 IgG receptor FcRn large subunit p51 Proteins 0.000 description 1
- 241000235058 Komagataella pastoris Species 0.000 description 1
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 1
- ONIBWKKTOPOVIA-BYPYZUCNSA-N L-Proline Chemical compound OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 0.000 description 1
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 1
- 101710175625 Maltose/maltodextrin-binding periplasmic protein Proteins 0.000 description 1
- UEZVMMHDMIWARA-UHFFFAOYSA-N Metaphosphoric acid Chemical compound OP(=O)=O UEZVMMHDMIWARA-UHFFFAOYSA-N 0.000 description 1
- 241000320412 Ogataea angusta Species 0.000 description 1
- 102000003992 Peroxidases Human genes 0.000 description 1
- 241000235350 Schizosaccharomyces octosporus Species 0.000 description 1
- 241000235347 Schizosaccharomyces pombe Species 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 125000003295 alanine group Chemical group N[C@@H](C)C(=O)* 0.000 description 1
- 239000012491 analyte Substances 0.000 description 1
- 210000004102 animal cell Anatomy 0.000 description 1
- 238000011953 bioanalysis Methods 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 238000011088 calibration curve Methods 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000003638 chemical reducing agent Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 210000004978 chinese hamster ovary cell Anatomy 0.000 description 1
- 239000000356 contaminant Substances 0.000 description 1
- OOTFVKOQINZBBF-UHFFFAOYSA-N cystamine Chemical compound CCSSCCN OOTFVKOQINZBBF-UHFFFAOYSA-N 0.000 description 1
- 229940099500 cystamine Drugs 0.000 description 1
- SUYVUBYJARFZHO-RRKCRQDMSA-N dATP Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@H]1C[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 SUYVUBYJARFZHO-RRKCRQDMSA-N 0.000 description 1
- SUYVUBYJARFZHO-UHFFFAOYSA-N dATP Natural products C1=NC=2C(N)=NC=NC=2N1C1CC(O)C(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 SUYVUBYJARFZHO-UHFFFAOYSA-N 0.000 description 1
- RGWHQCVHVJXOKC-SHYZEUOFSA-J dCTP(4-) Chemical compound O=C1N=C(N)C=CN1[C@@H]1O[C@H](COP([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O)[C@@H](O)C1 RGWHQCVHVJXOKC-SHYZEUOFSA-J 0.000 description 1
- HAAZLUGHYHWQIW-KVQBGUIXSA-N dGTP Chemical compound C1=NC=2C(=O)NC(N)=NC=2N1[C@H]1C[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 HAAZLUGHYHWQIW-KVQBGUIXSA-N 0.000 description 1
- NHVNXKFIZYSCEB-XLPZGREQSA-N dTTP Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)C1 NHVNXKFIZYSCEB-XLPZGREQSA-N 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 210000003527 eukaryotic cell Anatomy 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 108020001507 fusion proteins Proteins 0.000 description 1
- 102000037865 fusion proteins Human genes 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 229960003180 glutathione Drugs 0.000 description 1
- 238000004191 hydrophobic interaction chromatography Methods 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 229910017053 inorganic salt Inorganic materials 0.000 description 1
- 238000004255 ion exchange chromatography Methods 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 125000001360 methionine group Chemical group N[C@@H](CCSC)C(=O)* 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 210000001322 periplasm Anatomy 0.000 description 1
- 108040007629 peroxidase activity proteins Proteins 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 239000013600 plasmid vector Substances 0.000 description 1
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 1
- 210000001236 prokaryotic cell Anatomy 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000003753 real-time PCR Methods 0.000 description 1
- 101150079601 recA gene Proteins 0.000 description 1
- 108020003175 receptors Proteins 0.000 description 1
- 102000005962 receptors Human genes 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000004064 recycling Methods 0.000 description 1
- 230000010076 replication Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000002864 sequence alignment Methods 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 238000002849 thermal shift Methods 0.000 description 1
- 229940071127 thioglycolate Drugs 0.000 description 1
- CWERGRDVMFNCDR-UHFFFAOYSA-M thioglycolate(1-) Chemical compound [O-]C(=O)CS CWERGRDVMFNCDR-UHFFFAOYSA-M 0.000 description 1
- 230000007723 transport mechanism Effects 0.000 description 1
- 241001515965 unidentified phage Species 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Peptides Or Proteins (AREA)
Abstract
Description
本発明は、ヒト新生児（ｎｅｏｎａｔａｌ）Ｆｃレセプター（以下、ヒトＦｃＲｎという。）に由来し、免役グロブリンＧ（ＩｇＧ）やＩｇＧの定常領域であるＦｃ領域と他のタンパク質とを融合させた融合タンパク質（以下、Ｆｃ融合タンパク質という。）に対し結合親和性を有するＦｃ結合性タンパク質に関するものである。より詳しくは、タンパク質工学的手法を用いて生産性を向上させたＦｃ結合性タンパク質に関するものである。 The present invention is derived from a human neonatal Fc receptor (hereinafter referred to as human FcRn), and is a fusion protein obtained by fusing an Fc region which is a constant region of immunoglobulin G (IgG) or IgG with another protein (hereinafter referred to as human FcRn). Hereinafter, it relates to an Fc-binding protein having a binding affinity for (hereinafter referred to as Fc fusion protein). More specifically, it relates to Fc-binding proteins whose productivity has been improved by using protein engineering techniques.
ヒトＦｃＲｎは、ＩｇＧの受容体であり、ＩｇＧのＦｃ領域に結合する分子である。ヒトＦｃＲｎは、主要組織適合遺伝子複合体（ＭＨＣ）クラスＩ関連分子であり、重鎖（α鎖）と、β２ミクログロブリン（β鎖）により構成されている（非特許文献１）。ヒトＦｃＲｎのα鎖のアミノ酸配列（配列番号６）は、ＵｎｉＰｒｏｔ（Ａｃｃｅｓｓｉｏｎ ｎｕｍｂｅｒ：Ｐ５５８９９）などの公的データベースに公表されている。また、β鎖のアミノ酸配列（配列番号７）は、ＵｎｉＰｒｏｔ（Ａｃｃｅｓｓｉｏｎ ｎｕｍｂｅｒ：Ｐ６１７６９）に公表されている。 Human FcRn is a receptor for IgG and is a molecule that binds to the Fc region of IgG. Human FcRn is a major histocompatibility complex (MHC) class I-related molecule and is composed of a heavy chain (α chain) and β2 microglobulin (β chain) (Non-Patent Document 1). The amino acid sequence of the α chain of human FcRn (SEQ ID NO: 6) is published in public databases such as UniProt (Accession number: P55899). The amino acid sequence of the β chain (SEQ ID NO: 7) is published in UniProt (Accession number: P61769).
ヒトＦｃＲｎとＩｇＧ（Ｆｃ領域）の結合はｐＨ依存的であり、ｐＨ６．０−６．５で結合しｐＨ７．４以上で解離する。このｐＨ依存性はヒトＦｃＲｎの体内でのＩｇＧのリサイクリング機構や輸送機構に関与しており、ｐＨ依存的にヒトＦｃＲｎに結合・解離するＩｇＧおよびＦｃ融合タンパク質は体内寿命が長いことが知られている（非特許文献２）。ヒトＦｃＲｎのこの特徴を利用し、組換えヒトＦｃＲｎをリガンドとしたアフィニティーカラムを用いてヒトＩｇＧの体内寿命を評価する方法が知られている（非特許文献３）。 The binding between human FcRn and IgG (Fc region) is pH-dependent, binding at pH 6.0-6.5 and dissociating at pH 7.4 and above. This pH dependence is involved in the recycling mechanism and transport mechanism of IgG in the body of human FcRn, and it is known that IgG and Fc fusion proteins that bind to and dissociate from human FcRn in a pH-dependent manner have a long life in the body. (Non-Patent Document 2). Utilizing this feature of human FcRn, a method for evaluating the body life of human IgG using an affinity column using recombinant human FcRn as a ligand is known (Non-Patent Document 3).
前述した通り、組換えヒトＦｃＲｎはアフィニティーカラムのリガンドとして機能する特性を備えている。しかしながら、ヒトＦｃＲｎは熱やｐＨ変化などによりタンパク質変性が起こる傾向があった。そのため、特許文献１では、熱や酸に対する安定性が向上したヒトＦｃＲｎに由来したＦｃ結合性タンパク質が開示されている。 As mentioned above, recombinant human FcRn has the property of functioning as a ligand for an affinity column. However, human FcRn tends to undergo protein denaturation due to heat, pH change, or the like. Therefore, Patent Document 1 discloses an Fc-binding protein derived from human FcRn, which has improved stability against heat and acid.
本発明の課題は、特許文献１に開示のＦｃ結合性タンパク質よりもさらに生産性が向上したＦｃ結合性タンパク質、およびその製造方法を提供することにある。 An object of the present invention is to provide an Fc-binding protein having higher productivity than the Fc-binding protein disclosed in Patent Document 1, and a method for producing the same.
本発明者らは前記の課題を解決すべく鋭意検討した結果、Ｆｃ結合性タンパク質を構成するアミノ酸のうち特定の位置にあるアミノ酸を他の特定のアミノ酸に置換することにより、Ｆｃ結合性タンパク質の生産性が向上することを見出し、本発明を完成するに至った。 As a result of diligent studies to solve the above-mentioned problems, the present inventors have made an Fc-binding protein by substituting an amino acid at a specific position among the amino acids constituting the Fc-binding protein with another specific amino acid. We have found that productivity is improved, and have completed the present invention.
すなわち本発明は、以下の＜１＞から＜１１＞に記載の態様を包含する：
＜１＞
配列番号１に記載のアミノ酸配列の３４番目から４３１番目までのアミノ酸残基において少なくとも以下の（Ａ）から（Ｆ）のうち１以上のアミノ酸置換を有するアミノ酸配列を含む、Ｆｃ結合性タンパク質：
（Ａ）配列番号１の２１４番目のバリンがアスパラギン酸に置換、
（Ｂ）配列番号１の２３０番目のリジンがグルタミン酸に置換、
（Ｃ）配列番号１の２４２番目のリジンがグルタミン酸に置換、
（Ｄ）配列番号１の４００番目のリジンがグルタミン酸に置換、
（Ｅ）配列番号１の３０６番目のアスパラギンがアスパラギン酸に置換、
（Ｆ）配列番号１の３１５番目のセリンがスレオニンに置換。
That is, the present invention includes the aspects described in <1> to <11> below:
<1>
An Fc-binding protein comprising an amino acid sequence having at least one of the following amino acid substitutions (A) to (F) in the amino acid residues 34 to 431 of the amino acid sequence set forth in SEQ ID NO: 1.
(A) Valine at position 214 of SEQ ID NO: 1 is replaced with aspartic acid,
(B) The 230th lysine of SEQ ID NO: 1 is replaced with glutamic acid,
(C) The 242nd lysine of SEQ ID NO: 1 is replaced with glutamic acid,
(D) The 400th lysine of SEQ ID NO: 1 is replaced with glutamic acid,
(E) Asparagine at position 306 of SEQ ID NO: 1 is replaced with aspartic acid,
(F) Serine at position 315 of SEQ ID NO: 1 is replaced with threonine.
＜２＞
以下の（１）〜（３）の何れかに記載のタンパク質である、Ｆｃ結合性タンパク質：
（１）配列番号１に記載のアミノ酸配列の３４番目から４３１番目までのアミノ酸残基において、前記１以上のアミノ酸置換を有するアミノ酸配列を含む、Ｆｃ結合性タンパク質；
（２）配列番号１に記載のアミノ酸配列の３４番目から４３１番目までのアミノ酸残基において、前記１以上のアミノ酸置換を有し、さらに前記少なくとも１つのアミノ酸置換の位置以外の１若しくは数個の位置での１若しくは数個のアミノ酸残基の置換、欠失、挿入および付加のうち、いずれか１つ以上を有するアミノ酸配列を含む、Ｆｃ結合性タンパク質；
（３）配列番号１に記載のアミノ酸配列の３４番目から４３１番目までのアミノ酸残基において、前記１以上のアミノ酸置換を有するアミノ酸配列全体に対して８０％以上の相同性を有するアミノ酸配列であって、前記１以上のアミノ酸置換が残存したアミノ酸配列を含む、Ｆｃ結合性タンパク質。
<2>
Fc-binding protein, which is the protein according to any one of the following (1) to (3):
(1) An Fc-binding protein containing an amino acid sequence having one or more amino acid substitutions at the 34th to 431st amino acid residues of the amino acid sequence shown in SEQ ID NO: 1.
(2) At the amino acid residues 34 to 431 of the amino acid sequence shown in SEQ ID NO: 1, one or several amino acid residues other than the position of at least one amino acid substitution having one or more amino acid substitutions. An Fc-binding protein comprising an amino acid sequence having one or more of substitutions, deletions, insertions and additions of one or several amino acid residues at a position;
(3) The amino acid residues from the 34th to the 431st of the amino acid sequence shown in SEQ ID NO: 1 are amino acid sequences having 80% or more homology with the entire amino acid sequence having the amino acid substitution of 1 or more. An Fc-binding protein comprising an amino acid sequence in which one or more amino acid substitutions remain.
＜３＞
配列番号１に記載のアミノ酸配列のうち３４番目から４３１番目までのアミノ酸残基において前記（Ａ）から（Ｄ）のアミノ酸置換を有するアミノ酸配列を含む、＜１＞または＜２＞に記載のＦｃ結合性タンパク質。
<3>
Fc according to <1> or <2>, which comprises an amino acid sequence having the amino acid substitutions (A) to (D) in the amino acid residues 34 to 431 of the amino acid sequence shown in SEQ ID NO: 1. Binding protein.
＜４＞
さらに前記（Ｅ）および／または（Ｆ）のアミノ酸置換を有する、＜１＞〜＜３＞のいずれかに記載のＦｃ結合性タンパク質。
<4>
The Fc-binding protein according to any one of <1> to <3>, which further has the amino acid substitutions of (E) and / or (F).
＜５＞
配列番号２、配列番号３、配列番号４および配列番号５のうちいずれか１つに記載のアミノ酸配列における３４番目から４３１番目までのアミノ酸残基を含む、＜１＞〜＜４＞のいずれかに記載のＦｃ結合性タンパク質。
<5>
Any one of <1> to <4> containing the amino acid residues 34 to 431 in the amino acid sequence set forth in any one of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5. Fc-binding protein according to.
＜６＞
配列番号２、配列番号３、配列番号４および配列番号５のうちいずれか１つに記載のアミノ酸配列からなる、請求項１〜５のいずれかに記載のＦｃ結合性タンパク質。
<6>
The Fc-binding protein according to any one of claims 1 to 5, which comprises the amino acid sequence according to any one of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5.
＜７＞
＜１＞〜＜６＞のいずれかに記載のＦｃ結合性タンパク質をコードするＤＮＡ。
<7>
A DNA encoding the Fc-binding protein according to any one of <1> to <6>.
＜８＞
＜７＞に記載のＤＮＡを含む発現ベクター。
<8>
An expression vector containing the DNA according to <7>.
＜９＞
＜８＞に記載の発現ベクターで宿主を形質転換して得られる形質転換体。
<9>
A transformant obtained by transforming a host with the expression vector according to <8>.
＜１０＞
宿主が大腸菌（Ｅｓｃｈｅｒｉｃｈｉａ ｃｏｌｉ）である、＜９＞に記載の形質転換体。
<10>
The transformant according to <9>, wherein the host is Escherichia coli.
＜１１＞
＜９＞または＜１０＞に記載の形質転換体を培養することにより＜１＞〜＜６＞のいずれかに記載のＦｃ結合性タンパク質を生産する工程、および第１工程で生産されたＦｃ結合性タンパク質を回収する工程、の２つの工程を含む、Ｆｃ結合性タンパク質の製造方法。
<11>
The step of producing the Fc-binding protein according to any one of <1> to <6> by culturing the transformant according to <9> or <10>, and the Fc binding produced in the first step. A method for producing an Fc-binding protein, which comprises two steps of recovering the sex protein.
以下、本発明を詳細に説明する。 Hereinafter, the present invention will be described in detail.
本発明のＦｃ結合性タンパク質における、アミノ酸置換の基準となるＦｃ結合性タンパク質ＦｃＲｎ＿ｍ７のアミノ酸配列（配列番号１）は、特開２０１８−１８３０８７号公報で開示のＦｃ結合性タンパク質ＦｃＲｎ＿ｍ７のアミノ酸配列と一致する。なお、配列番号１において、その１番目のメチオニンから２６番目のアラニンまでがＭａｌＥシグナルペプチドであり、２７番目のリジンから３３番目のグリシンまでがリンカー配列であり、３４番目のイソロイシンから１３２番目のメチオニンまでがヒトＦｃＲｎβ鎖のβ２ミクログロブリン領域［配列番号７（Ｕｎｉｐｒｏｔ登録番号：Ｐ６１７６９）の２１番目から１１９番目の領域］であり、１３３番目のグリシンから１５７番目のセリンまでがＧＳリンカー配列であり、１５８番目のアラニンから４３１番目のセリンまでが７アミノ酸置換を有するヒトＦｃＲｎα鎖の細胞外領域［配列番号６（Ｕｎｉｐｒｏｔ登録番号：Ｐ５５８９９）の２４番目から２９７番目の領域のアミノ酸配列に相当し、かつそのうち（配列番号６）の７１番目のシステインがアルギニンに置換、７８番目のアスパラギンがアスパラギン酸に置換、１５１番目のグリシンがアスパラギン酸に置換、１９２番目のアルギニンがロイシンに置換、１９６番目のアスパラギンがアスパラギン酸に置換、２３２番目のグルタミンがロイシンに置換および２９５番目のリジンがグルタミン酸に置換された配列］であり、４３２番目から４３７番目まではポリヒスチジン配列である。 The amino acid sequence (SEQ ID NO: 1) of the Fc-binding protein FcRn_m7, which is a reference for amino acid substitution, in the Fc-binding protein of the present invention matches the amino acid sequence of the Fc-binding protein FcRn_m7 disclosed in JP-A-2018-183087. To do. In SEQ ID NO: 1, from the 1st methionine to the 26th alanine is the MalE signal peptide, from the 27th lysine to the 33rd glycine is the linker sequence, and from the 34th isoleucine to the 132nd methionine. Is the β2 microglobulin region of the human FcRnβ chain [the 21st to 119th regions of SEQ ID NO: 7 (Uniprot registration number: P61769)], and the 133rd to 157th serine is the GS linker sequence. The extracellular region of the human FcRnα chain from alanine at position 158 to serine at position 431 has a 7-amino acid substitution [corresponding to the amino acid sequence of the 24th to 297th regions of SEQ ID NO: 6 (Uniprot registration number: P55899), and Of these, the 71st cysteine of (SEQ ID NO: 6) was replaced with arginine, the 78th aspartic acid was replaced with aspartic acid, the 151st glycine was replaced with aspartic acid, the 192nd arginine was replaced with leucine, and the 196th aspartic acid was replaced. A sequence in which aspartic acid is substituted, glycine at position 232 is substituted with leucine, and lysine at position 295 is substituted with glutamic acid], and positions 432 to 437 are polyhistidine sequences.
なお「高生産性」とは、野生型（配列番号６および７に記載のヒトＦｃＲｎに比べて生産性が向上していることを意味してよく、好ましくは配列番号１に記載のヒトＦｃＲｎに比べて生産性が向上していることを意味してよい。ここで、向上するとは、比較対象（野生型または特許文献１に記載のヒトＦｃＲｎ）と比べて生産性が５％以上、好ましくは１０％以上、より好ましくは１５％以上高くなることをいう。 The "high productivity" may mean that the productivity is improved as compared with the wild type (human FcRn set forth in SEQ ID NOs: 6 and 7, and preferably the human FcRn set forth in SEQ ID NO: 1). It may mean that the productivity is improved as compared with the above. Here, the improvement means that the productivity is 5% or more, preferably 5% or more, as compared with the comparison target (wild type or human FcRn described in Patent Document 1). It means that it is increased by 10% or more, more preferably 15% or more.
本発明のＦｃ結合性タンパク質は、
配列番号１に記載のアミノ酸配列の３４番目から４３１番目までのアミノ酸残基において少なくとも以下の（Ａ）から（Ｆ）のうち１以上のアミノ酸置換を有するアミノ酸残基を含む、Ｆｃ結合性タンパク質：
（Ａ）配列番号１の２１４番目のバリンがアスパラギン酸に置換、
（Ｂ）配列番号１の２３０番目のリジンがグルタミン酸に置換、
（Ｃ）配列番号１の２４２番目のリジンがグルタミン酸に置換、
（Ｄ）配列番号１の４００番目のリジンがグルタミン酸に置換、
（Ｅ）配列番号１の３０６番目のアスパラギンがアスパラギン酸に置換、
（Ｆ）配列番号１の３１５番目のセリンがスレオニンに置換。
The Fc-binding protein of the present invention is
An Fc-binding protein comprising an amino acid residue having at least one of the following amino acid substitutions (A) to (F) in the amino acid residues 34 to 431 of the amino acid sequence set forth in SEQ ID NO: 1.
(A) Valine at position 214 of SEQ ID NO: 1 is replaced with aspartic acid,
(B) The 230th lysine of SEQ ID NO: 1 is replaced with glutamic acid,
(C) The 242nd lysine of SEQ ID NO: 1 is replaced with glutamic acid,
(D) The 400th lysine of SEQ ID NO: 1 is replaced with glutamic acid,
(E) Asparagine at position 306 of SEQ ID NO: 1 is replaced with aspartic acid,
(F) Serine at position 315 of SEQ ID NO: 1 is replaced with threonine.
前記態様の具体例として、以下の（１）〜（３）に示す、Ｆｃ結合性タンパク質が挙げられる。
（１）配列番号１に記載のアミノ酸配列の３４番目から４３１番目までのアミノ酸残基において、前記１以上のアミノ酸置換を有するアミノ酸配列を含む、Ｆｃ結合性タンパク質。
（２）配列番号１に記載のアミノ酸配列の３４番目から４３１番目までのアミノ酸残基において、前記１以上のアミノ酸置換を有し、さらに前記少なくとも１つのアミノ酸置換の位置以外の１若しくは数個の位置での１若しくは数個のアミノ酸残基の置換、欠失、挿入および付加のうち、いずれか１つ以上を有するアミノ酸配列を含む、Ｆｃ結合性タンパク質。
（３）配列番号１に記載のアミノ酸配列の３４番目から４３１番目までのアミノ酸残基において、前記１以上のアミノ酸置換を有するアミノ酸配列全体に対して８０％以上の相同性を有するアミノ酸配列であって、前記１以上のアミノ酸置換が残存したアミノ酸配列を含む、Ｆｃ結合性タンパク質。
Specific examples of the above-described embodiment include Fc-binding proteins shown in the following (1) to (3).
(1) An Fc-binding protein comprising an amino acid sequence having one or more amino acid substitutions in the amino acid residues 34 to 431 of the amino acid sequence set forth in SEQ ID NO: 1.
(2) At the amino acid residues 34 to 431 of the amino acid sequence shown in SEQ ID NO: 1, one or several amino acid residues other than the position of at least one amino acid substitution having one or more amino acid substitutions. An Fc-binding protein comprising an amino acid sequence having any one or more of substitutions, deletions, insertions and additions of one or several amino acid residues at a position.
(3) The amino acid residues from the 34th to the 431st of the amino acid sequence shown in SEQ ID NO: 1 are amino acid sequences having 80% or more homology with the entire amino acid sequence having the amino acid substitution of 1 or more. An Fc-binding protein comprising an amino acid sequence in which one or more amino acid substitutions remain.
本発明のＦｃ結合性タンパク質は、少なくとも前記置換（Ｃ）が生じていることが好ましく、前記置換（Ａ）から置換（Ｄ）が全て生じていることが更に好ましい。 The Fc-binding protein of the present invention preferably has at least the above-mentioned substitution (C), and more preferably all the substitutions (D) from the above-mentioned substitution (A).
本発明のＦｃ結合性タンパク質は、前記の置換（Ａ）から置換（Ｄ）に記載の全てのアミノ酸置換に加え、前記の置換（Ｅ）および／または置換（Ｆ）に記載のアミノ酸置換が生じることで、形質転換体による生産性（発現量）がさらに向上する。 In the Fc-binding protein of the present invention, in addition to all the amino acid substitutions described in the above substitutions (A) to (D), the amino acid substitutions described in the above substitutions (E) and / or substitutions (F) occur. As a result, the productivity (expression level) of the transformant is further improved.
本発明のＦｃ結合性タンパク質は、前記の置換（Ａ）から置換（Ｆ）に記載の６つのアミノ酸置換が生じることで、前記の配列番号１に記載のＦｃ結合性タンパク質ＦｃＲｎ＿ｍ７に比べ、熱安定性が向上する。 The Fc-binding protein of the present invention is thermally stable as compared with the Fc-binding protein FcRn_m7 described in SEQ ID NO: 1 by causing the six amino acid substitutions described in the substitution (F) from the substitution (A). Improves sex.
本発明のＦｃ結合性タンパク質の一例として、
（ａ）配列番号１に記載のアミノ酸配列のうち３４番目から４３１番目までのアミノ酸配列を含み、かつ当該３４番目から４３１番目までのアミノ酸配列において置換（Ａ）、置換（Ｂ）、置換（Ｃ）および置換（Ｄ）に記載のアミノ酸置換が生じているＦｃ結合性タンパク質（配列番号２に記載のアミノ酸配列のうち３４番目から４３１番目までのアミノ酸配列を含むＦｃ結合性タンパク質）、
（ｂ）配列番号１に記載のアミノ酸配列のうち３４番目から４３１番目までのアミノ酸配列を含み、かつ当該３４番目から４３１番目までのアミノ酸配列において置換（Ａ）、置換（Ｂ）、置換（Ｃ）、置換（Ｄ）および置換（Ｅ）に記載のアミノ酸置換が生じているＦｃ結合性タンパク質（配列番号３に記載のアミノ酸配列のうち３４番目から４３１番目までのアミノ酸配列を含むＦｃ結合性タンパク質）、
（ｃ）配列番号１に記載のアミノ酸配列のうち３４番目から４３１番目までのアミノ酸配列を含み、かつ当該３４番目から４３１番目までのアミノ酸配列において置換（Ａ）、置換（Ｂ）、置換（Ｃ）、置換（Ｄ）および置換（Ｆ）に記載のアミノ酸置換が生じているＦｃ結合性タンパク質（配列番号４に記載のアミノ酸配列のうち３４番目から４３１番目までのアミノ酸配列を含むＦｃ結合性タンパク質）、および
（ｄ）配列番号１に記載のアミノ酸配列のうち３４番目から４３１番目までのアミノ酸配列を含み、かつ当該３４番目から４３１番目までのアミノ酸配列において置換（Ａ）、置換（Ｂ）、置換（Ｃ）、置換（Ｄ）、置換（Ｅ）および置換（Ｆ）に記載のアミノ酸置換が生じているＦｃ結合性タンパク質（配列番号５に記載のアミノ酸配列のうち３４番目から４３１番目までのアミノ酸配列を含むＦｃ結合性タンパク質）、
が挙げられる。
As an example of the Fc-binding protein of the present invention,
(A) Of the amino acid sequences shown in SEQ ID NO: 1, the amino acid sequences from 34th to 431st are included, and the amino acid sequences from 34th to 431st are substituted (A), substituted (B), substituted (C). ) And the Fc-binding protein in which the amino acid substitution shown in Substitution (D) has occurred (Fc-binding protein containing the amino acid sequences 34 to 431 of the amino acid sequences shown in SEQ ID NO: 2),
(B) Of the amino acid sequences shown in SEQ ID NO: 1, the amino acid sequences from 34th to 431st are included, and the amino acid sequences from 34th to 431st are substituted (A), substituted (B), substituted (C). ), Substitution (D) and Fc-binding protein containing the amino acid substitutions shown in Substitution (E) (Fc-binding protein containing the amino acid sequences 34 to 431 of the amino acid sequences shown in SEQ ID NO: 3). ),
(C) Of the amino acid sequences shown in SEQ ID NO: 1, the amino acid sequences from 34th to 431st are included, and substitutions (A), substitutions (B), and substitutions (C) are made in the amino acid sequences from 34th to 431st. ), Substitution (D) and Fc-binding protein in which the amino acid substitution described in Substitution (F) occurs (Fc-binding protein containing the 34th to 431st amino acid sequences of the amino acid sequences shown in SEQ ID NO: 4). ), And (d) the amino acid sequences from 34th to 431st among the amino acid sequences shown in SEQ ID NO: 1, and substitutions (A), substitutions (B), in the amino acid sequences from 34th to 431st. Fc-binding proteins in which the amino acid substitutions described in Substitution (C), Substitution (D), Substitution (E) and Substitution (F) have occurred (from the 34th to 431st amino acid sequences shown in SEQ ID NO: 5). Fc-binding protein containing amino acid sequence),
Can be mentioned.
本発明のＦｃ結合性タンパク質は、ＩｇＧのＦｃ領域結合性を有している限り、そのＮ末端側および／またはＣ末端側に、夾雑物質存在下の溶液から分離する際に有用な付加的なアミノ酸配列を有していてもよい。前記付加的なアミノ酸配列としては、ポリヒスチジン配列、グルタチオンＳ−トランスフェラーゼ、マルトース結合タンパク質、セルロース結合性ドメイン、ｍｙｃタグ、ＦＬＡＧタグ等が挙げられる。これらの付加的なアミノ酸配列の中では、ニッケルキレートアフィニティークロマトグラフィーによる精製が容易に行える点で、ポリヒスチジン配列を含むオリゴペプチドであることが好ましい。 As long as the Fc-binding protein of the present invention has Fc region-binding property of IgG, it is useful for separating from the solution in the presence of contaminants on its N-terminal side and / or C-terminal side. It may have an amino acid sequence. Examples of the additional amino acid sequence include a polyhistidine sequence, glutathione S-transferase, maltose-binding protein, cellulose-binding domain, myc tag, FLAG tag and the like. Among these additional amino acid sequences, oligopeptides containing a polyhistidine sequence are preferable because they can be easily purified by nickel chelate affinity chromatography.
さらに、本発明のＦｃ結合性タンパク質は、ＩｇＧのＦｃ領域結合性を有している限り、そのＮ末端側および／またはＣ末端側に、本発明のＦｃ結合性タンパク質をクロマトグラフィー用の支持体等の担体に固定化する際に有用な、システインまたはリジンを含むオリゴペプチドからなる付加的なアミノ酸配列（以下、担体固定化用タグと呼ぶ。）を有していても良い。 Furthermore, as long as the Fc-binding protein of the present invention has the Fc region-binding property of IgG, the Fc-binding protein of the present invention is placed on its N-terminal side and / or C-terminal side as a support for chromatography. It may have an additional amino acid sequence consisting of an oligopeptide containing cysteine or lysine (hereinafter, referred to as a carrier immobilization tag), which is useful for immobilization on a carrier such as.
加えて、本発明のＦｃ結合性タンパク質のＮ末端側には、宿主での効率的な発現を促すためのシグナルペプチドを付加してもよい。宿主が大腸菌（Ｅｓｃｈｅｒｉｃｈｉａ ｃｏｌｉ）の場合における前記シグナルペプチドとしては、ＰｅｌＢ、ＤｓｂＡ、ＭａｌＥ、ＴｏｒＴ等といったペリプラズムにタンパク質を分泌させるシグナルペプチドを例示することができる。 In addition, a signal peptide for promoting efficient expression in the host may be added to the N-terminal side of the Fc-binding protein of the present invention. As the signal peptide when the host is Escherichia coli, a signal peptide such as PelB, DsbA, MalE, TorT, etc. that causes periplasm to secrete a protein can be exemplified.
本明細書において、「１もしくは数個のアミノ酸残基の置換、欠失、挿入または付加」、ならびに「アミノ酸残基の置換、欠失、挿入および付加のうち、いずれか１つ以上」とは、タンパク質の立体構造におけるアミノ酸残基の位置やアミノ酸残基の種類によっても異なるが、例えば、１から５０個のアミノ酸残基の置換、欠失、挿入または付加であってよく、好ましくは１から４０個、より好ましくは１から３０個、更に好ましくは１から２０個、特に好ましくは１から１０個のアミノ酸残基の置換、欠失、挿入または付加であってよい。また本明細書における「置換」、「欠失」、「挿入」および「付加」には、遺伝子が由来する生物（微生物も包含する）の個体差、種の違いなどに基づく、天然にも生じ得る変異（ｍｕｔａｎｔまたはｖａｒｉａｎｔ）も含まれる。 As used herein, the term "substitution, deletion, insertion or addition of one or several amino acid residues" and "one or more of substitution, deletion, insertion and addition of amino acid residues" are used. , Depending on the position of the amino acid residue in the three-dimensional structure of the protein and the type of amino acid residue, for example, it may be substitution, deletion, insertion or addition of 1 to 50 amino acid residues, preferably from 1. It may be 40, more preferably 1 to 30, more preferably 1 to 20, and particularly preferably 1 to 10 amino acid residues substitution, deletion, insertion or addition. In addition, "substitution", "deletion", "insertion" and "addition" in the present specification naturally occur based on individual differences, species differences, etc. of the organism (including microorganisms) from which the gene is derived. The resulting mutation (mutant or variant) is also included.
前記（２）におけるアミノ酸配列の相同性は７０％以上であってよく、好ましくは８０％以上、より好ましくは８５％以上、更に好ましくは９０％以上であってよい。 The homology of the amino acid sequence in (2) may be 70% or more, preferably 80% or more, more preferably 85% or more, and further preferably 90% or more.
本明細書において、アミノ酸配列の「相同性」は、アミノ酸配列の「同一性」と同義である。ここで、アミノ酸配列の「同一性（相同性）」とは、比較すべき２つのアミノ酸配列のアミノ酸残基ができるだけ多く一致するように両アミノ酸配列を整列させ、一致したアミノ酸残基数を全アミノ酸残基数で除したものを百分率で表したものである。上記整列の際には、必要に応じ、比較する２つの配列の一方又は双方に適宜ギャップを挿入する。このような配列の整列化方法は、特に限定されないが、例えばＢＬＡＳＴ、ＦＡＳＴＡ、ＣＬＵＳＴＡＬ Ｗ等の周知の配列比較プログラムを用いて行なうことができる。ギャップが挿入される場合、上記全アミノ酸残基数は、１つのギャップを１つのアミノ酸残基として数えた残基数となる。このようにして数えた全アミノ酸残基数が、比較する２つの配列間で異なる場合には、配列同一性（％）は、長い方の配列の全アミノ酸残基数で、一致したアミノ酸残基数を除して算出される。 As used herein, "homology" of an amino acid sequence is synonymous with "identity" of an amino acid sequence. Here, "identity (homosphere)" of amino acid sequences means that both amino acid sequences are aligned so that the amino acid residues of the two amino acid sequences to be compared match as much as possible, and the total number of matched amino acid residues is totaled. It is expressed as a percentage by dividing by the number of amino acid residues. At the time of the above alignment, if necessary, a gap is appropriately inserted in one or both of the two sequences to be compared. Such a sequence alignment method is not particularly limited, but can be performed using, for example, a well-known sequence comparison program such as BLAST, FASTA, or Clustal W. When a gap is inserted, the total number of amino acid residues is the number of residues counted as one amino acid residue. When the total number of amino acid residues counted in this way differs between the two sequences to be compared, the sequence identity (%) is the total number of amino acid residues in the longer sequence, and the matching amino acid residues. Calculated by dividing the number.
本発明のＦｃ結合性タンパク質は、両アミノ酸の物理的性質と化学的性質またはそのどちらかが類似したアミノ酸間で置換する保守的置換をさらに有してもよい。保守的置換は、Ｆｃ結合性タンパク質に限らず一般に、置換が生じているものと置換が生じていないものとの間でタンパク質の機能が維持されることが当業者において知られている。保守的置換の一例としては、グリシンとアラニン間、アスパラギン酸とグルタミン酸間、セリンとプロリン間、またはグルタミン酸とアラニン間に生じる置換があげられる（タンパク質の構造と機能，メディカル・サイエンス・インターナショナル社，９，２００５）。 The Fc-binding proteins of the present invention may further have conservative substitutions in which the physical and / or chemical properties of both amino acids are similar. Conservative substitutions are not limited to Fc-binding proteins and are generally known to those skilled in the art to maintain the function of the protein between those with and without substitution. Examples of conservative substitutions include substitutions that occur between glycine and alanine, aspartic acid and glutamic acid, serine and proline, or between glutamic acid and alanine (Protein Structure and Function, Medical Science International, Inc., 9). , 2005).
本発明のＦｃ結合性タンパク質の一例として、そのＮ末端側にＭａｌＥシグナルペプチドが付加され、かつそのＣ末端側にポリヒスチジンが付加された、
（ｅ）配列番号２のアミノ酸配列からなるＦｃ結合性タンパク質、
（ｆ）配列番号３のアミノ酸配列からなるＦｃ結合性タンパク質、
（ｇ）配列番号４のアミノ酸配列からなるＦｃ結合性タンパク質、
（ｈ）配列番号５のアミノ酸配列からなるＦｃ結合性タンパク質、
が挙げられる。
As an example of the Fc-binding protein of the present invention, a MalE signal peptide was added to the N-terminal side thereof, and polyhistidine was added to the C-terminal side thereof.
(E) An Fc-binding protein consisting of the amino acid sequence of SEQ ID NO: 2.
(F) Fc-binding protein consisting of the amino acid sequence of SEQ ID NO: 3.
(G) Fc-binding protein consisting of the amino acid sequence of SEQ ID NO: 4,
(H) An Fc-binding protein consisting of the amino acid sequence of SEQ ID NO: 5.
Can be mentioned.
次に、本発明のＦｃ結合性タンパク質をコードするＤＮＡ（以下、本発明のＤＮＡとする。）および本発明のＤＮＡを含有する発現ベクター（以下、本発明の発現ベクターとする。）について説明する。 Next, a DNA encoding the Fc-binding protein of the present invention (hereinafter referred to as the DNA of the present invention) and an expression vector containing the DNA of the present invention (hereinafter referred to as the expression vector of the present invention) will be described. ..
本発明のＤＮＡは、（Ｉ）Ｐｏｌｙｍｅｒａｓｅ Ｃｈａｉｎ Ｒｅａｃｔｉｏｎ（ＰＣＲ）法といったＤＮＡ増幅法を利用してヒトゲノムＤＮＡのＦｃＲｎをコードする領域をもとに改変して作製する方法、（ＩＩ）ヒトＦｃＲｎのアミノ酸配列（配列番号６および配列番号７）から塩基配列に変換し、当該塩基配列を含むＤＮＡを人工的に作製し、それをさらにＤＮＡ増幅法を利用し改変して作製する方法、または、（ＩＩＩ）例えば配列番号２から配列番号５に記載のアミノ酸配列を塩基配列に変換し、当該塩基配列を含むＤＮＡを人工的に作製する方法等により得ることができる。これらの方法において、アミノ酸配列から塩基配列に変換する際には、本発明のＦｃ結合性タンパク質の生産に利用する宿主におけるコドンの使用頻度を考慮することが好ましい。一例として、大腸菌を宿主として利用する場合、アルギニンではＡＧＡ、ＡＧＧ、ＣＧＧまたはＣＧＡが、イソロイシンではＡＴＡが、ロイシンではＣＴＡが、グリシンではＧＧＡが、プロリンではＣＣＣが、それぞれ使用頻度が少ないコドン（レアコドン）であるため、これらのコドンを避けるように変換することが好ましい。コドンの使用頻度の解析は公的データベース（例えば、かずさＤＮＡ研究所のホームページにあるＣｏｄｏｎ Ｕｓａｇｅ Ｄａｔａｂａｓｅ、ｈｔｔｐ：／／ｗｗｗ．ｋａｚｕｓａ．ｏｒ．ｊｐ／ｃｏｄｏｎ／、アクセス日：２０１９年５月３０日）を利用することによっても可能である。 The DNA of the present invention is prepared by modifying a region encoding FcRn of human genomic DNA by using a DNA amplification method such as (I) Polymerase Chain Reaction (PCR) method, (II) Amino acid of human FcRn. A method of converting a sequence (SEQ ID NO: 6 and SEQ ID NO: 7) into a base sequence, artificially preparing a DNA containing the base sequence, and further modifying it using a DNA amplification method, or (III). ) For example, it can be obtained by converting the amino acid sequences set forth in SEQ ID NO: 2 to SEQ ID NO: 5 into a base sequence and artificially producing a DNA containing the base sequence. In these methods, when converting from an amino acid sequence to a base sequence, it is preferable to consider the frequency of use of codons in the host used for the production of the Fc-binding protein of the present invention. As an example, when Escherichia coli is used as a host, AGA, AGG, CGG or CGA for arginine, ATA for isoleucine, CTA for leucine, GGA for glycine, and CCC for proline are infrequently used codons (rare codons). ), Therefore it is preferable to convert so as to avoid these codons. Analysis of codon usage frequency is performed in a public database (for example, Codon User Database on the homepage of Kazusa DNA Research Institute, http: //www.kazusa.or.jp/codon/, access date: May 30, 2019). It is also possible by using.
ＤＮＡ増幅法を用いて本発明のＤＮＡを作製する際は、エラープローンＰＣＲ法を用いた変異導入法を利用することができる。エラープローンＰＣＲ法における反応条件は、ＤＮＡに所望の変異を導入できる条件であれば特に限定はなく、例えば、基質である４種類のデオキシヌクレオチド（ｄＡＴＰ／ｄＴＴＰ／ｄＣＴＰ／ｄＧＴＰ）の濃度を不均一にし、ＭｎＣｌ２を０．０１から１０ｍＭ、好ましくは０．１から１ｍＭの濃度でＰＣＲ反応液に添加してＰＣＲを行なうことができる。 When producing the DNA of the present invention using the DNA amplification method, a mutation introduction method using the error prone PCR method can be used. The reaction conditions in the error-prone PCR method are not particularly limited as long as they can introduce a desired mutation into DNA. For example, the concentrations of four types of deoxynucleotides (dATP / dTTP / dCTP / dGTP) as substrates are non-uniform. Then, MnCl 2 can be added to the PCR reaction solution at a concentration of 0.01 to 10 mM, preferably 0.1 to 1 mM to carry out PCR.
本発明のＤＮＡとして、具体的には、配列番号２のアミノ酸配列をコードする配列番号８の塩基配列からなるＤＮＡ、配列番号３のアミノ酸配列をコードする配列番号９の塩基配列からなるＤＮＡ、配列番号４のアミノ酸配列をコードする配列番号１０の塩基配列からなるＤＮＡ、および配列番号５のアミノ酸配列をコードする配列番号１１の塩基配列からなるＤＮＡを例示することができる。 Specifically, the DNA of the present invention includes a DNA consisting of the nucleotide sequence of SEQ ID NO: 8 encoding the amino acid sequence of SEQ ID NO: 2, a DNA consisting of the nucleotide sequence of SEQ ID NO: 9 encoding the amino acid sequence of SEQ ID NO: 3, and a sequence. Examples thereof include a DNA consisting of the base sequence of SEQ ID NO: 10 encoding the amino acid sequence of No. 4 and a DNA consisting of the base sequence of SEQ ID NO: 11 encoding the amino acid sequence of SEQ ID NO: 5.
本発明のＤＮＡを用いて宿主を形質転換する場合、本発明のＤＮＡそのものを用いてもよいが、ベクター（例えば、原核細胞や真核細胞の形質転換に通常用いるバクテリオファージ、コスミドまたはプラスミド等）の適切な位置に本発明のＤＮＡを挿入した発現ベクター（本発明の発現ベクター）を用いると、安定した形質転換が実施できる点で好ましい。ここで、適切な位置とは、ベクターの複製機能、所望の抗生物質マーカー、および伝達性に関わる領域を破壊しない位置を意味する。また、ベクターに本発明のＤＮＡを挿入する際は、発現に必要なプロモータといった機能性ＤＮＡに連結される状態で挿入することが好ましい。 When transforming a host with the DNA of the present invention, the DNA of the present invention itself may be used, but a vector (for example, bacteriophage, cosmid or plasmid usually used for transformation of prokaryotic cells or eukaryotic cells). It is preferable to use an expression vector (expression vector of the present invention) in which the DNA of the present invention is inserted at an appropriate position in the above, because stable transformation can be carried out. As used herein, the appropriate position means a position that does not disrupt the replication function of the vector, the desired antibiotic marker, and the region involved in transmissibility. When inserting the DNA of the present invention into a vector, it is preferable to insert it in a state of being linked to a functional DNA such as a promoter required for expression.
本発明の発現ベクターとして使用するベクターは、宿主内で安定に存在し複製できるものであれば特に制限はなく、例えば大腸菌を宿主とする場合、ｐＥＴベクター、ｐＵＣベクター、ｐＴｒｃベクター、ｐＣＤＦベクター、ｐＢＢＲベクター等のプラスミドベクターが例示できる。また本発明において使用するプロモータとしては、例えば大腸菌を宿主とする場合、ｔｒｐプロモータ、ｔａｃプロモータ、ｔｒｃプロモータ、ｌａｃプロモータ、Ｔ７プロモータ、ｒｅｃＡプロモータ、ｌｐｐプロモータ、さらにはλファージのλＰＬプロモータ、λＰＲプロモータ等を挙げることができる。 The vector used as the expression vector of the present invention is not particularly limited as long as it exists stably in the host and can be replicated. For example, when using Escherichia coli as the host, the pET vector, pUC vector, pTrc vector, pCDF vector, pBBR A plasmid vector such as a vector can be exemplified. Further, as the promoter used in the present invention, for example, when Escherichia coli is used as a host, a trp promoter, a tac promoter, a trc promoter, a lac promoter, a T7 promoter, a recA promoter, a lpp promoter, a λ phage λPL promoter, a λPR promoter, etc. Can be mentioned.
本発明のＦｃ結合性タンパク質を生産可能な形質転換体（以下、本発明の形質転換体とする。）は、本発明の発現ベクターを用いて宿主を形質転換することで得ることができる。本発明の形質転換体として使用する宿主に特に制限はなく、一例として、動物細胞（ＣＨＯ細胞、ＨＥＫ細胞、Ｈｅｌａ細胞、ＣＯＳ細胞等）、酵母（Ｓａｃｃｈａｒｏｍｙｃｅｓ ｃｅｒｅｖｉｓｉａｅ、Ｐｉｃｈｉａ ｐａｓｔｏｒｉｓ、Ｈａｎｓｅｎｕｌａ ｐｏｌｙｍｏｒｐｈａ、Ｓｃｈｉｚｏｓａｃｃｈａｒｏｍｙｃｅｓ ｊａｐｏｎｉｃｕｓ、Ｓｃｈｉｚｏｓａｃｃｈａｒｏｍｙｃ ｅｓ ｏｃｔｏｓｐｏｒｕｓ、Ｓｃｈｉｚｏｓａｃｃｈａｒｏｍｙｃｅｓ ｐｏｍｂｅ等）、昆虫細胞（Ｓｆ９、Ｓｆ２１等）、大腸菌［ＪＭ１０９株、ＢＬ２１（ＤＥ３）株、Ｗ３１１０株等］、枯草菌等があげられる。なお、遺伝子工学に関する実験が容易な点および生産性の点で、大腸菌を宿主として用いることが好ましい。本発明の発現ベクターを用いて宿主を形質転換するには、当業者が通常用いる方法で行えばよく、例えば、宿主として大腸菌［ＪＭ１０９株、ＢＬ２１（ＤＥ３）株、Ｗ３１１０株等］を選択する場合には、公知の文献（例えば、Ｍｏｌｅｃｕｌａｒ Ｃｌｏｎｉｎｇ，Ｃｏｌｄ Ｓｐｒｉｎｇ Ｈａｒｂｏｒ Ｌａｂｏｒａｔｏｒｙ，２５６，１９９２）に記載の方法等により形質転換すればよい。 A transformant capable of producing the Fc-binding protein of the present invention (hereinafter referred to as a transformant of the present invention) can be obtained by transforming a host with the expression vector of the present invention. The host used as the transformant of the present invention is not particularly limited, and as an example, animal cells (CHO cells, HEK cells, Hela cells, COS cells, etc.), yeasts (Saccharomyces cerevisiae, Pichia pastoris, Hansenula polymorpha, Schizos). Examples thereof include Schizosaccharomyces octosporus, Schizosaccharomyces pombe, etc.), insect cells (Sf9, Sf21, etc.), Escherichia coli [JM109 strain, BL21 (DE3) strain, W3110 strain, etc.], Saccharomyces cerevisiae, and the like. It is preferable to use Escherichia coli as a host in terms of ease of experiments related to genetic engineering and productivity. To transform a host using the expression vector of the present invention, a method usually used by those skilled in the art may be used. For example, when Escherichia coli [JM109 strain, BL21 (DE3) strain, W3110 strain, etc.] is selected as the host. Can be transformed by the method described in known literature (for example, Molecular Cloning, Cold Spring Host Laboratory, 256, 1992).
本発明の形質転換体から本発明の発現ベクターを調製するには、形質転換に用いた宿主に適した方法で、本発明の形質転換体から本発明の発現ベクターを抽出し、調製すればよい。例えば本発明の形質転換体の宿主が大腸菌の場合、形質転換体を培養して得られる培養物からアルカリ抽出法またはＱＩＡｐｒｅｐ Ｓｐｉｎ Ｍｉｎｉｐｒｅｐ ｋｉｔ（キアゲン製）等の市販の抽出キットを用いて調製すればよい。 In order to prepare the expression vector of the present invention from the transformant of the present invention, the expression vector of the present invention may be extracted from the transformant of the present invention and prepared by a method suitable for the host used for the transformation. .. For example, when the host of the transformant of the present invention is Escherichia coli, it can be prepared from the culture obtained by culturing the transformant using an alkaline extraction method or a commercially available extraction kit such as QIAprep Spin Miniprep kit (manufactured by Qiagen). Good.
次に、本発明のＦｃ結合性タンパク質の製造方法（以下、本発明の製造方法とする。）について説明する。本発明の製造方法は、本発明の形質転換体を培養することで本発明のＦｃ結合性タンパク質を生産する工程（以下、第１工程という。）、第１工程で生産された本発明のＦｃ結合性タンパク質を培養物から回収する工程（以下、第２工程という。）の２つの工程を含む。なお本明細書において、培養物とは、第１工程において培養された本発明の形質転換体の細胞自体や細胞分泌物のほか、培養に用いた培地等も含まれる。 Next, the method for producing the Fc-binding protein of the present invention (hereinafter referred to as the production method of the present invention) will be described. The production method of the present invention is a step of producing the Fc-binding protein of the present invention by culturing the transformant of the present invention (hereinafter referred to as the first step), and the Fc of the present invention produced in the first step. It includes two steps of recovering the binding protein from the culture (hereinafter referred to as a second step). In the present specification, the culture includes not only the cells themselves and cell secretions of the transformant of the present invention cultured in the first step, but also the medium used for the culture and the like.
本発明の製造方法における第１工程では、形質転換体をその培養に適した培地で培養すればよい。例えば、宿主として大腸菌を用いた場合、必要な栄養源を補ったＴｅｒｒｉｆｉｃ Ｂｒｏｔｈ（ＴＢ）培地、Ｌｕｒｉａ−Ｂｅｒｔａｎｉ（ＬＢ）培地等を使用することが好ましい。本発明の発現ベクターが薬剤耐性遺伝子を含む場合、その遺伝子に対応した薬剤を培地に添加して第１工程を実施すれば、形質転換体の選択的増殖が可能となり、例えば、当該発現ベクターがカナマイシン耐性遺伝子を含んでいる場合は、培地にカナマイシンを添加すればよい。また、培地には、炭素、窒素および無機塩供給源の他に、適当な栄養源を添加してもよく、所望により、グルタチオン、システイン、シスタミン、チオグリコレートおよびジチオスレイトールからなる群から選択される一種類以上の還元剤を含んでもよい。さらにグリシン等の前記形質転換体から培養液へのタンパク質分泌を促す試薬を添加してもよく、具体的には、宿主が大腸菌の場合、培地に対してグリシンを２％（ｗ／ｖ）以下で添加すると好ましい。培養温度は利用する宿主に関して一般的に知られた温度であればよく、例えば宿主が大腸菌である場合、１０℃から４０℃、好ましくは２０℃から３７℃であり、本発明のＦｃ結合性タンパク質の特性により選択すればよい。培地のｐＨは宿主が大腸菌の場合、ｐＨ６．８からｐＨ７．４、好ましくはｐＨ７．０前後である。本発明の発現ベクターに誘導性のプロモータを導入した場合は、本発明のＦｃ結合性タンパク質が良好に製造可能な条件下で培地に誘導剤を添加してその発現を誘導すればよい。誘導剤としてはｉｓｏｐｒｏｐｙｌ−β−Ｄ−ｔｈｉｏｇａｌａｃｔｏｐｙｒａｎｏｓｉｄｅ（ＩＰＴＧ）を例示することができ、その添加濃度は０．００５から１．０ｍＭの範囲、好ましくは０．０１から０．５ｍＭの範囲である。ＩＰＴＧ添加による発現誘導は、利用する宿主に関して一般的に知られた条件で行なえばよい。 In the first step in the production method of the present invention, the transformant may be cultured in a medium suitable for the culture. For example, when Escherichia coli is used as a host, it is preferable to use a Terrific Broth (TB) medium supplemented with necessary nutrient sources, a Luria-Bertani (LB) medium, or the like. When the expression vector of the present invention contains a drug resistance gene, if a drug corresponding to the gene is added to the medium and the first step is carried out, the transformant can be selectively grown. For example, the expression vector can be used. If the kanamycin resistance gene is contained, kanamycin may be added to the medium. In addition to carbon, nitrogen and inorganic salt sources, suitable nutrient sources may be added to the medium, optionally selected from the group consisting of glutathione, cysteine, cystamine, thioglycolate and dithiothreitol. It may contain one or more reducing agents to be used. Further, a reagent such as glycine that promotes protein secretion from the transformant to the culture medium may be added. Specifically, when the host is Escherichia coli, glycine is 2% (w / v) or less based on the medium. It is preferable to add in. The culture temperature may be any temperature generally known for the host to be used, for example, when the host is Escherichia coli, it is 10 ° C. to 40 ° C., preferably 20 ° C. to 37 ° C., and the Fc-binding protein of the present invention. It may be selected according to the characteristics of. When the host is Escherichia coli, the pH of the medium is pH 6.8 to pH 7.4, preferably around pH 7.0. When an inducible promoter is introduced into the expression vector of the present invention, an inducer may be added to the medium under conditions under which the Fc-binding protein of the present invention can be satisfactorily produced to induce its expression. As the inducer, isopropanol-β-D-thiogalactopylanoside (IPTG) can be exemplified, and the addition concentration thereof is in the range of 0.005 to 1.0 mM, preferably in the range of 0.01 to 0.5 mM. Expression induction by addition of IPTG may be performed under generally known conditions for the host to be used.
本発明の製造方法における第２工程では、第１工程で生産された本発明のＦｃ結合性タンパク質を培養物から一般的に知られた回収方法によって回収する。例えば本発明のＦｃ結合性タンパク質が培養液中に分泌生産される場合は細胞を遠心分離操作によって分離し、得られる培養上清から本発明のＦｃ結合性タンパク質を回収すればよく、細胞内（原核生物においてはペリプラズムも含む）に発現する場合は、遠心分離操作により細胞を集めた後、酵素処理剤や界面活性剤等を添加する等により細胞を破砕し、細胞破砕液から回収すればよい。 In the second step in the production method of the present invention, the Fc-binding protein of the present invention produced in the first step is recovered from the culture by a generally known recovery method. For example, when the Fc-binding protein of the present invention is secreted and produced in a culture medium, the cells may be separated by a centrifugation operation, and the Fc-binding protein of the present invention may be recovered from the obtained culture supernatant. When expressed in prokaryotic organisms (including periplasmum), the cells may be collected by centrifugation, then the cells may be disrupted by adding an enzyme treatment agent, a surfactant, or the like, and then recovered from the cell disruption solution. ..
本発明の製造方法により回収された本発明のＦｃ結合性タンパク質の純度を向上したい場合には、当該技術分野において公知の方法を用いればよく、一例として、液体クロマトグラフィーを用いた分離精製法を挙げることができる。液体クロマトグラフィーとしては、イオン交換クロマトグラフィー、疎水性相互作用クロマトグラフィー、ゲルろ過クロマトグラフィー、アフィニティークロマトグラフィー等を使用することが好ましく、これらのクロマトグラフィーを組み合わせて行なうことがより好ましい。また、前記クロマトグラフィーにより精製した本発明のＦｃ結合性タンパク質の純度は当該技術分野において公知の方法を用いて調べればよく、一例として、ＳＤＳ（Ｓｏｄｉｕｍ ｄｏｄｅｃｙｌ ｓｕｌｆａｔｅ）ポリアクリルアミドゲル電気泳動（ＳＤＳ−ＰＡＧＥ）法やゲルろ過クロマトグラフィー法を挙げることができる。 When it is desired to improve the purity of the Fc-binding protein of the present invention recovered by the production method of the present invention, a method known in the art may be used, and as an example, a separation and purification method using liquid chromatography may be used. Can be mentioned. As the liquid chromatography, it is preferable to use ion exchange chromatography, hydrophobic interaction chromatography, gel filtration chromatography, affinity chromatography and the like, and it is more preferable to perform these chromatography in combination. Further, the purity of the Fc-binding protein of the present invention purified by the chromatography may be examined by using a method known in the art, and as an example, SDS (Sodium dodecyl sulfate) polyacrylamide gel electrophoresis (SDS-PAGE). ) Method and gel filtration chromatography method can be mentioned.
本発明のＦｃ結合性タンパク質のＩｇＧへの結合親和性の評価は、Ｅｎｚｙｍｅ−ｌｉｎｋｅｄ ｉｍｍｕｎｏｓｏｒｂｅｎｔ ａｓｓａｙ（ＥＬＩＳＡ）法や表面プラズモン共鳴法等を用いて測定すればよい。 The evaluation of the binding affinity of the Fc-binding protein of the present invention to IgG may be measured by using an enzyme-linked immunosorbent assay (ELISA) method, a surface plasmon resonance method, or the like.
本発明のＦｃ結合性タンパク質は、特開２０１８−１８３０８７号公報で開示されている既知のＦｃ結合性タンパク質と比較し、生産性が向上している。特開２０１８−１８３０８７号公報で開示されている既知のＦｃ結合性タンパク質と同様に、本発明のＦｃ結合性タンパク質はＩｇＧまたはＦｃ融合タンパク質を分離するための吸着剤のリガンドとして有用であり、生産性の向上により工業的生産に適している。 The Fc-binding protein of the present invention has improved productivity as compared with the known Fc-binding protein disclosed in JP-A-2018-183087. Similar to the known Fc-binding proteins disclosed in JP-A-2018-183087, the Fc-binding proteins of the present invention are useful as ligands for adsorbents for separating IgG or Fc fusion proteins and are produced. It is suitable for industrial production due to its improved properties.
以下、実施例、比較例および参考例をあげて本発明をさらに詳細に説明するが、本発明はこれらに限定されるものではない。 Hereinafter, the present invention will be described in more detail with reference to Examples, Comparative Examples and Reference Examples, but the present invention is not limited thereto.
比較例１ Ｆｃ結合性タンパク質ＦｃＲｎ＿ｍ７の製造と評価
（１）Ｆｃ結合性タンパク質ＦｃＲｎ＿ｍ７の製造
特開２０１８−１８３０８７号公報では、Ｆｃ結合性タンパク質ＦｃＲｎ＿ｍ７が開示されている。Ｆｃ結合性タンパク質ＦｃＲｎ＿ｍ７のアミノ酸配列を配列番号１に示す。
（１−１）発現ベクターｐＥＴ−ＦｃＲｎ＿ｍ７の作製
特開２０１８−１８３０８７号公報で開示されている発現ベクターｐＥＴ−ＦｃＲｎ＿ｍ７を、当該公報に記載された方法で作製した。この発現ベクターｐＥＴ−ＦｃＲｎ＿ｍ７を用いて大腸菌ＢＬ２１（ＤＥ３）を形質転換し、組換え大腸菌ＢＬ２１（ＤＥ３）／ｐＥＴ−ＦｃＲｎ＿ｍ７を得た。
（１−２）Ｆｃ結合性タンパク質ＦｃＲｎ＿ｍ７の製造
前記の組換え大腸菌ＢＬ２１（ＤＥ３）／ｐＥＴ−ＦｃＲｎ＿ｍ７を３０μｇ／ｍＬのカナマイシンを添加したＬＢ培地（１０ｇ／Ｌ ｔｒｙｐｔｏｎｅ，５ｇ／Ｌ Ｙｅａｓｔ ｅｘｔｒａｃｔおよび５ｇ／Ｌ ＮａＣｌ）に接種し、３７℃で一晩振盪することで前培養を行った。前培養液をそれぞれ３０μｇ／ｍＬのカナマイシンを添加した１５０ｍＬのＴＢ培地（２４ｇ／Ｌ Ｙｅａｓｔ ｅｘｔｒａｃｔ、１２ｇ／Ｌ ｔｒｙｐｔｏｎｅ、９．４ｇ／Ｌ Ｋ２ＨＰＯ４、２．２ｇ／Ｌ ＫＨ２ＰＯ４および４ｍＬ／Ｌ Ｇｌｙｃｅｒｏｌ）に接種し、３７℃で振盪培養した。培養液の濁度（Ｏ．Ｄ．６００）が凡そ０．６になったところで、培養温度を２０℃に切り替え、終濃度０．０５ｍＭのＩＰＴＧを添加し約１８時間振盪培養した。遠心操作により培養液から得られた菌体から、ＢｕｇＢｕｓｔｅｒ Ｐｒｏｔｅｉｎ ｅｘｔｒａｃｔｉｏｎ ｋｉｔ（メルク製）を用いて可溶性タンパク質抽出液を回収した。可溶性タンパク質抽出液からのＦｃ結合性タンパク質ＦｃＲｎ＿ｍ７の精製は、Ｈｉｓ・Ｂｉｎｄ Ｒｅｓｉｎ（メルク製）を用いたニッケルキレートアフィニティークロマトグラフィーにより行った。精製済みのＦｃ結合性タンパク質ＦｃＲｎ＿ｍ７が高純度であることをＳＤＳ−ＰＡＧＥにより確認した。精製済みのＦｃ結合性タンパク質ＦｃＲｎ＿ｍ７のタンパク質濃度はＭｉｃｒｏ ＢＣＡ Ｐｒｏｔｅｉｎ Ａｓｓａｙ Ｋｉｔ（サーモフィッシャーサイエンティフィック製）を用いて定量した。
（２）Ｆｃ結合性タンパク質ＦｃＲｎ＿ｍ７の生産性評価
Ｆｃ結合性タンパク質ＦｃＲｎ＿ｍ７の生産性評価として、培養液１Ｌあたりの発現タンパク質量（発現量）を評価した。
Comparative Example 1 Production and Evaluation of Fc-Binding Protein FcRn_m7 (1) Production of Fc-Binding Protein FcRn_m7 Japanese Patent Application Laid-Open No. 2018-183087 discloses Fc-binding protein FcRn_m7. The amino acid sequence of the Fc-binding protein FcRn_m7 is shown in SEQ ID NO: 1.
(1-1) Preparation of Expression Vector pET-FcRn_m7 The expression vector pET-FcRn_m7 disclosed in JP-A-2018-183087 was prepared by the method described in the publication. Escherichia coli BL21 (DE3) was transformed with this expression vector pET-FcRn_m7 to obtain recombinant Escherichia coli BL21 (DE3) / pET-FcRn_m7.
(1-2) Production of Fc-binding protein FcRn_m7 LB medium (10 g / L tryptone, 5 g / L Yeast extract and 5 g /) supplemented with 30 μg / mL kanamycin of the above-mentioned recombinant Escherichia coli BL21 (DE3) / pET-FcRn_m7. L NaCl) was inoculated and precultured by shaking at 37 ° C. overnight. 150 mL TB medium (24 g / L Yeast extract, 12 g / L tryptone, 9.4 g / L K 2 HPO 4 , 2.2 g / L KH 2 PO 4 and 4 mL, respectively, to which 30 μg / mL of canamycin was added to the preculture solution. / L Glycerol) was inoculated and cultured with shaking at 37 ° C. When the turbidity (OD600) of the culture solution reached about 0.6, the culture temperature was switched to 20 ° C., IPTG having a final concentration of 0.05 mM was added, and the cells were shake-cultured for about 18 hours. A soluble protein extract was recovered from the cells obtained from the culture broth by centrifugation using a Bug Buster Protein extraction kit (manufactured by Merck). Purification of the Fc-binding protein FcRn_m7 from the soluble protein extract was performed by nickel chelate affinity chromatography using His-Bind Resin (manufactured by Merck). It was confirmed by SDS-PAGE that the purified Fc-binding protein FcRn_m7 had high purity. The protein concentration of the purified Fc-binding protein FcRn_m7 was quantified using the Micro BCA Protein Assay Kit (manufactured by Thermo Fisher Scientific).
(2) Productivity evaluation of Fc-binding protein FcRn_m7 As a productivity evaluation of Fc-binding protein FcRn_m7, the amount of expressed protein (expression level) per 1 L of culture solution was evaluated.
比較例１の（１−１）に記載の組換え大腸菌ＢＬ２１（ＤＥ３）／ｐＥＴ−ＦｃＲｎ＿ｍ７を３０μｇ／ｍＬのカナマイシンを添加したＬＢ培地に接種し、３７℃で一晩振盪することで前培養を行った。前培養液をそれぞれ３０μｇ／ｍＬのカナマイシンを添加した３０ｍＬのＴＢ培地に接種し（１００ｍＬバッフル付フラスコを使用）、３７℃で振盪培養した。培養液の濁度（Ｏ．Ｄ．６００）が凡そ０．６になったところで、培養温度を２０℃に切り替え、終濃度０．０５ｍＭのＩＰＴＧを添加し約１８時間振盪培養した。遠心操作により培養液（２ｍＬ）から得られた菌体から、ＢｕｇＢｕｓｔｅｒ Ｐｒｏｔｅｉｎ ｅｘｔｒａｃｔｉｏｎ ｋｉｔ（メルク製）を用いて可溶性タンパク質抽出液を回収した。この可溶性タンパク質抽出液を適宜希釈し、ＳＤＳ−ＰＡＧＥに供した。比較例１の（１）で作製した精製済みのＦｃ結合性タンパク質ＦｃＲｎ＿ｍ７を幾つかの濃度に調整し（１ウェルあたり１．５ｎｇから１５ｎｇの範囲）、濃度の標準としてＳＤＳ−ＰＡＧＥに供した。ＳＤＳ−ＰＡＧＥ後、ゲルからメンブレンへタンパク質の転写を行ない、ウェスタンブロッティングによる検出を行った。検出抗体はＨｏｒｓｅｒａｄｉｓｈ ｐｅｒｏｘｉｄａｓｅ標識抗Ｈｉｓ抗体、発色基質はＴＭＢを使用した。濃度既知である精製済みのＦｃ結合性タンパク質ＦｃＲｎ＿ｍ７のバンドシグナル強度から検量線を作成し、可溶性タンパク質抽出液のＦｃ結合性タンパク質ＦｃＲｎ＿ｍ７の濃度を定量した。本濃度を基に培養液１Ｌあたりの発現タンパク質量（発現量）を算出した。 The recombinant Escherichia coli BL21 (DE3) / pET-FcRn_m7 according to (1-1) of Comparative Example 1 was inoculated into an LB medium supplemented with 30 μg / mL kanamycin, and precultured by shaking at 37 ° C. overnight. went. The precultures were inoculated into 30 mL TB medium supplemented with 30 μg / mL kanamycin (using a flask with a 100 mL baffle) and cultured with shaking at 37 ° C. When the turbidity (OD600) of the culture solution reached about 0.6, the culture temperature was switched to 20 ° C., IPTG having a final concentration of 0.05 mM was added, and the cells were shake-cultured for about 18 hours. Soluble protein extract was recovered from the cells obtained from the culture broth (2 mL) by centrifugation using the Bug Buster Protein extraction kit (manufactured by Merck). This soluble protein extract was appropriately diluted and subjected to SDS-PAGE. The purified Fc-binding protein FcRn_m7 prepared in Comparative Example 1 (1) was adjusted to several concentrations (range 1.5 ng to 15 ng per well) and subjected to SDS-PAGE as a standard concentration. After SDS-PAGE, the protein was transferred from the gel to the membrane and detected by Western blotting. Horseradish peroxidase-labeled anti-His antibody was used as the detection antibody, and TMB was used as the color-developing substrate. A calibration curve was prepared from the band signal intensity of the purified Fc-binding protein FcRn_m7 whose concentration was known, and the concentration of the Fc-binding protein FcRn_m7 in the soluble protein extract was quantified. The amount of expressed protein (expression amount) per 1 L of the culture solution was calculated based on this concentration.
その結果、Ｆｃ結合性タンパク質ＦｃＲｎ＿ｍ７の発現量は４５ｍｇ／Ｌであった（表１）。 As a result, the expression level of the Fc-binding protein FcRn_m7 was 45 mg / L (Table 1).
（３）Ｆｃ結合性タンパク質ＦｃＲｎ＿ｍ７の抗体に対する結合親和性評価
Ｆｃ結合性タンパク質ＦｃＲｎ＿ｍ７のヒト抗体に対する結合親和性評価を表面プラズモン共鳴法により行った。具体的には、Ｂｉａｃｏｒｅ Ｔ１００（Ｔ２００ Ｓｅｎｓｉｔｉｖｉｔｙ Ｅｎｈａｎｃｅｄ）機器（ＧＥヘルスケア製）を用い、アナライトをヒト抗体製剤（人免疫グロブリン グロブリン筋注、日本薬局方製）、固相をＦｃ結合性タンパク質ＦｃＲｎ＿ｍ７としてカイネティクス解析を行った。センサーチップはニトリロ三酢酸（ＮＴＡ）をコートしたセンサーチップ（Ｓｅｎｓｏｒ Ｃｈｉｐ ＮＴＡ、ＧＥヘルスケア製）を使用し、ＮＴＡにニッケルを固定した後、精製済みのＦｃ結合性タンパク質ＦｃＲｎ＿ｍ７をセンサーチップに固定した（Ｆｃ結合性タンパク質ＦｃＲｎ＿ｍ７のＣ末端側のポリヒスチジンとニッケルの結合を利用）。抗体結合性の測定にはｐＨ６．０緩衝液（６７ｍＭ リン酸緩衝液、１５０ｍＭ 塩化ナトリウム、０．０５％ Ｔｗｅｅｎ２０）を用い、カイネティクス解析はシングルサイクルカイネティクス法により行った。測定条件は表２に示す。解析はＢｉａｃｏｒｅ Ｔ１００（Ｔ２００ Ｓｅｎｓｉｔｉｖｉｔｙ Ｅｎｈａｎｃｅｄ）機器に付属の解析ソフト（Ｂｉａｃｏｒｅ Ｔ２００ Ｅｖａｌｕａｔｉｏｎ Ｓｏｆｔｗａｒｅ）を用いて行い、アナライト濃度（Ｃ）に対する平衡値（Ｒｅｑ）のプロットから解離定数（ＫＤ）の値を算出した。解離定数（ＫＤ）の値は小さいほど結合親和性が高いことを示す。
(3) Evaluation of binding affinity of Fc-binding protein FcRn_m7 for antibody The binding affinity of Fc-binding protein FcRn_m7 for human antibody was evaluated by the surface plasmon resonance method. Specifically, using a Biacore T100 (T200 Sensitivity Enhanced) device (manufactured by GE Healthcare), the analysis is a human antibody preparation (human immunoglobulin globulin intramuscular injection, manufactured by the Japanese Pharmacopoeia), and the solid phase is an Fc-binding protein FcRn_m7. Kinetics analysis was performed as. As the sensor chip, a sensor chip coated with nitrilotriacetic acid (NTA) (Sensor Chip NTA, manufactured by GE Healthcare) was used, nickel was fixed to NTA, and then the purified Fc-binding protein FcRn_m7 was fixed to the sensor chip. (Using the bond between polyhistidine and nickel on the C-terminal side of the Fc-binding protein FcRn_m7). The antibody binding was measured using a pH 6.0 buffer (67 mM phosphate buffer, 150 mM sodium chloride, 0.05% Tween 20), and the kinetics analysis was performed by the single cycle kinetics method. The measurement conditions are shown in Table 2. Analysis was performed using a Biacore T100 (T200 Sensitivity Enhanced) analysis software (Biacore T200 Evaluation Software) supplied with the device, the value of the plot from the dissociation constant of the equilibrium value (Req) (K D) for the analyte concentration (C) Calculated. The value of the dissociation constant (K D) shows that the higher the binding affinity less.
Ｆｃ結合性タンパク質ＦｃＲｎ＿ｍ７のヒト抗体に対する解離定数（ＫＤ）の値は１０±４μＭ（ｎ＝３）であった。 The value of the dissociation constant for human antibody Fc binding protein FcRn_m7 (K D) was 10 ± 4μM (n = 3) .
（４）Ｆｃ結合性タンパク質ＦｃＲｎ＿ｍ７の熱安定性評価
（４−１）サンプル調製
比較例１の（１）で製造した精製済みのＦｃ結合性タンパク質ＦｃＲｎ＿ｍ７の溶媒を、Ａｍｉｃｏｎ Ｕｌｔｒａ遠心式フィルター（メルク製）を使用してＤ−ＰＢＳ（−）緩衝液に置換し、タンパク質濃度を定量した（紫外線吸収法；Ａ２８０＝１．０を１．０ｍｇ／ｍＬとした）。
（４−２）熱安定性評価
熱安定性の評価はリアルタイムＰＣＲ装置ＱｕａｎｔＳｔｕｄｉｏ３（サーモフィッシャーサイエンティフィック製）を使用してＴｈｅｒｍａｌ ｓｈｉｆｔ Ａｓｓａｙ法により行った。タンパク質濃度は０．２５から０．５ｍｇ／ｍＬの範囲、蛍光色素はＳＹＰＲＯ Ｏｒａｎｇｅ［終濃度０．５％（ｖ／ｖ）］、昇温温度は３０℃から８５℃、昇温速度は０．０２５℃／ｓの測定条件とした。変性中点の算出にはＱｕａｎｔＳｔｕｄｉｏ Ｄｅｓｉｇｎ＆Ａｎａｌｙｓｉｓ Ｓｏｆｔｗａｒｅ ｖｅｒ．１．２（サーモフィッシャーサイエンティフィック製）を使用した。
(4) Thermal stability evaluation of Fc-binding protein FcRn_m7 (4-1) Sample preparation The solvent of the purified Fc-binding protein FcRn_m7 produced in (1) of Comparative Example 1 was used as an Amicon Ultra centrifugal filter (manufactured by Merck). ) Was replaced with D-PBS (−) buffer to quantify the protein concentration (ultraviolet absorption method; A280 = 1.0 was 1.0 mg / mL).
(4-2) Evaluation of thermal stability The evaluation of thermal stability was performed by the Thermal shift Assay method using a real-time PCR device QuantStudio3 (manufactured by Thermo Fisher Scientific). The protein concentration is in the range of 0.25 to 0.5 mg / mL, the fluorescent dye is SYPRO Orange [final concentration 0.5% (v / v)], the temperature rise temperature is 30 ° C. to 85 ° C., and the temperature rise rate is 0. The measurement condition was 025 ° C./s. For the calculation of the midpoint of denaturation, Quant Studio Design & Analysis Software ver. 1.2 (manufactured by Thermo Fisher Scientific) was used.
Ｆｃ結合性タンパク質ＦｃＲｎ＿ｍ７の変性中点は６０±２℃（ｎ＝２）であった。 The midpoint of denaturation of the Fc-binding protein FcRn_m7 was 60 ± 2 ° C. (n = 2).
実施例１ 変異導入Ｆｃ結合性タンパク質からのＦｃ結合性タンパク質候補のスクリーニング
（１）Ｆｃ結合性タンパク質へのランダム変異導入とランダム変異体ライブリーの作製
比較例１の（１−１）に記載の発現ベクターｐＥＴ−ＦｃＲｎ＿ｍ７を鋳型として、配列番号１２および配列番号１３からなる各オリゴヌクレオチドをＰＣＲプライマーとしてエラープローンＰＣＲを行なった。エラープローンＰＣＲは、特開２０１８−５０６１６号公報で開示されている方法で実施した。得られたＰＣＲ産物をｍ７−ＥＰとした。ＰＣＲ産物ｍ７−ＥＰは、制限酵素ＮｃｏＩおよびＨｉｎｄＩＩＩで消化し、あらかじめ同制限酵素で消化した特開２０１８−１８３０８７号公報で開示されている発現ベクターｐＥＴＭａｌＥとライゲーション反応を行った。このライゲーション産物を用いて大腸菌ＢＬ２１（ＤＥ３）を形質転換し、３０μｇ／ｍＬのカナマイシンを添加したＬＢ寒天培地（１０ｇ／Ｌ ｔｒｙｐｔｏｎｅ，５ｇ／Ｌ Ｙｅａｓｔ ｅｘｔｒａｃｔ，５ｇ／Ｌ ＮａＣｌ，１５ｇ／Ｌ Ａｇａｒ）で培養（３７℃で約１８時間）後、ＬＢ寒天培地上に形成されたコロニーをランダム変異体ライブリー（形質転換体）とした。
（２）Ｆｃ結合性タンパク質候補のスクリーニング
（２−１）発現培養
前記（１）のランダム変異体ライブリー（形質転換体）および比較例１の（１−１）に記載の組換え大腸菌ＢＬ２１（ＤＥ３）／ｐＥＴ−ＦｃＲｎ＿ｍ７（比較対照として）をそれぞれ３０μｇ／ｍＬのカナマイシンを添加したＬＢ培地４００μＬに接種し、９６穴ディープウェルプレートを用いて、３７℃で一晩振盪することで前培養を行った。各前培養液１５μＬは３０μｇ／ｍＬのカナマイシンを添加したＴＢ培地５００μＬに接種し、９６穴ディープウェルプレートを用いて、３７℃で振盪培養した。２時間後に培養温度を２０℃に切り替え、終濃度０．０５ｍＭのＩＰＴＧおよび０．３％（ｗ／ｖ）のグリシンをそれぞれ添加後、約１８時間振盪培養した。各培養液に対して遠心操作を行い、発現した組換えタンパク質を含む培養上清を得た。この培養上清サンプルに対して加熱処理（４６℃で１０分間）を行い、評価サンプルとした。
（２−２）抗体結合性評価を指標としたスクリーニング
抗体結合性評価として以下のＥＬＩＳＡ法を実施した。
Example 1 Screening of Fc-binding protein candidates from mutation-introduced Fc-binding protein (1) Random mutation introduction into Fc-binding protein and preparation of random mutant primer. Error prone PCR was performed using the expression vector pET-FcRn_m7 as a template and each oligonucleotide consisting of SEQ ID NO: 12 and SEQ ID NO: 13 as a PCR primer. Error prone PCR was carried out by the method disclosed in JP-A-2018-50616. The obtained PCR product was designated as m7-EP. The PCR product m7-EP was digested with restriction enzymes NcoI and HindIII, and ligated with the expression vector pETMalE disclosed in JP-A-2018-183087, which had been previously digested with the restriction enzymes. Escherichia coli BL21 (DE3) was transformed with this ligation product and LB agar medium (10 g / L typetone, 5 g / L Yeast extract, 5 g / L NaCl, 15 g / L Agar) supplemented with 30 μg / mL canamycin. After culturing (about 18 hours at 37 ° C.), colonies formed on LB agar medium were designated as random mutant Escherichia coli (transformant).
(2) Screening of Fc-binding protein candidates (2-1) Expression culture The recombinant Escherichia coli BL21 (1-1) according to the random mutant library (transformant) of (1) above and Comparative Example 1 (1-1). DE3) / pET-FcRn_m7 (as a control) was inoculated into 400 μL of LB medium supplemented with 30 μg / mL kanamycin, respectively, and precultured by shaking overnight at 37 ° C. using a 96-well deepwell plate. It was. Each 15 μL of preculture was inoculated into 500 μL of TB medium supplemented with 30 μg / mL kanamycin, and cultured with shaking at 37 ° C. using a 96-well deep well plate. After 2 hours, the culture temperature was switched to 20 ° C., IPTG having a final concentration of 0.05 mM and 0.3% (w / v) of glycine were added, and the cells were cultured with shaking for about 18 hours. Each culture solution was centrifuged to obtain a culture supernatant containing the expressed recombinant protein. This culture supernatant sample was heat-treated (at 46 ° C. for 10 minutes) to prepare an evaluation sample.
(2-2) Screening using antibody binding evaluation as an index The following ELISA method was performed as antibody binding evaluation.
ヒトＩｇＧを含む抗体製剤（人免疫グロブリン グロブリン筋注、日本薬局方製）をＤ−ＰＢＳ（−）緩衝液で１０μｇ／ｍＬの濃度に調整後、それを９６穴マイクロプレート（ＭａｘｉＳｏｒｐ、Ｎｕｎｃ社製）の各ウェルに１００μＬ／ｗｅｌｌ添加し、４℃で１８時間静置しＩｇＧを固定化した。固定化終了後、各ウェルの溶液を捨て、２％（ｗ／ｖ）のＳｋｉｍ Ｍｉｌｋ（Ｂｅｃｔｏｎ，Ｄｉｃｋｉｎｓｏｎ ａｎｄ Ｃｏｍｐａｎｙ製）を含むＤ−ＰＢＳ（−）を２００μＬ／ｗｅｌｌ添加し、３０℃で２時間静置しブロッキングを行った。洗浄緩衝液［５０ｍＭ リン酸緩衝液（ｐＨ６．０）、１５０ｍＭ 塩化ナトリウム、０．０５％（ｗ／ｖ）Ｔｗｅｅｎ ２０］を用いて各ウェルを洗浄した。各ウェルに５０μＬ／ｗｅｌｌの評価サンプル調整緩衝液［２００ｍＭ リン酸緩衝液（ｐＨ５．８）、３００ｍＭ 塩化ナトリウム］を添加した。続いて、前記（２−１）で調製した評価サンプルを５０μＬ／ｗｅｌｌ添加し、混合後、３０℃で１時間静置し、固定化ＩｇＧと反応させた。反応後、洗浄緩衝液を用いて各ウェルを洗浄し、Ｈｏｒｓｅｒａｄｉｓｈ Ｐｅｒｏｘｉｄａｓｅ標識抗Ｈｉｓ−Ｔａｇ抗体試薬（ＢＥＴＨＹＬ社製）［２％（ｗ／ｖ）のＳｋｉｍ Ｍｉｌｋを含む洗浄緩衝液で希釈］を各ウェルに添加し、３０℃で１時間静置した。洗浄緩衝液を用いて各ウェルを洗浄し、５０μＬ／ｗｅｌｌのＴＭＢ Ｐｅｒｏｘｉｄａｓｅ Ｓｕｂｓｔｒａｔｅ（セラケアライフサイエンシーズ社製）を各ウェルに添加した。５０μＬ／ｗｅｌｌの１Ｍ リン酸を添加することで発色反応を止め、マイクロプレートリーダー（Ｔｅｃａｎ製）にて４５０ｎｍの吸光度を測定した。 After adjusting the antibody preparation containing human IgG (human immunoglobulin globulin intramuscular injection, manufactured by Nippon Pharmacy Co., Ltd.) to a concentration of 10 μg / mL with D-PBS (-) buffer, it is prepared into a 96-well microplate (MaxiSorp, manufactured by Nunc). ), 100 μL / well was added, and the mixture was allowed to stand at 4 ° C. for 18 hours to immobilize IgG. After completion of immobilization, discard the solution in each well, add 200 μL / well of D-PBS (-) containing 2% (w / v) of Skim Milk (manufactured by Becton, Dickinson and Company), and add at 30 ° C. for 2 hours. It was allowed to stand and blocked. Each well was washed with wash buffer [50 mM phosphate buffer (pH 6.0), 150 mM sodium chloride, 0.05% (w / v) Tween 20]. A 50 μL / well evaluation sample preparation buffer [200 mM phosphate buffer (pH 5.8), 300 mM sodium chloride] was added to each well. Subsequently, 50 μL / well of the evaluation sample prepared in (2-1) above was added, and after mixing, the mixture was allowed to stand at 30 ° C. for 1 hour to react with immobilized IgG. After the reaction, each well was washed with a wash buffer and diluted with Horseradish Peroxidase-labeled anti-His-Tag antibody reagent (BETHYL) [diluted with wash buffer containing 2% (w / v) Skim Milk]. It was added to the wells and allowed to stand at 30 ° C. for 1 hour. Each well was washed with wash buffer and 50 μL / well TMB Peroxidase Substrate (manufactured by Ceracare Life Sciences) was added to each well. The color reaction was stopped by adding 50 μL / well of 1 M phosphoric acid, and the absorbance at 450 nm was measured with a microplate reader (manufactured by Tecan).
ＥＬＩＳＡ法の４５０ｎｍの吸光度は、抗体結合性を有する組換えタンパク質量と相関する。組換え大腸菌ＢＬ２１（ＤＥ３）／ｐＥＴ−ＦｃＲｎ＿ｍ７（比較対照：Ｆｃ結合性タンパク質ＦｃＲｎ＿ｍ７を生産）の培養上清と比較し、顕著に吸光度が増加した形質転換体をランダム変異体ライブリーから選抜した。選抜されたそれぞれの形質転換体から発現ベクターを抽出し、塩基配列を解析した。
（３）Ｆｃ結合性タンパク質候補
前記（２）のスクリーニングの結果、以下のＦｃ結合性タンパク質を発現可能な形質転換体を取得した。
（３−１）Ｆｃ結合性タンパク質ＦｃＲｎ＿ｍ８ａ
Ｆｃ結合性タンパク質ＦｃＲｎ＿ｍ８ａ（アミノ酸配列：配列番号１４）をコードする塩基配列（配列番号１５）を含む発現ベクターｐＥＴ−ＦｃＲｎ＿ｍ８ａ、およびそれを含有する形質転換体である組換え大腸菌ＢＬ２１（ＤＥ３）／ＦｃＲｎ＿ｍ８ａを取得した。
（３−２）Ｆｃ結合性タンパク質ＦｃＲｎ＿ｍ８ｂ
Ｆｃ結合性タンパク質ＦｃＲｎ＿ｍ８ｂ（アミノ酸配列：配列番号１６）をコードする塩基配列（配列番号１７）を含む発現ベクターｐＥＴ−ＦｃＲｎ＿ｍ８ｂ、およびそれを含有する形質転換体である組換え大腸菌ＢＬ２１（ＤＥ３）／ＦｃＲｎ＿ｍ８ｂを取得した。
（３−３）Ｆｃ結合性タンパク質ＦｃＲｎ＿ｍ１１
Ｆｃ結合性タンパク質ＦｃＲｎ＿ｍ１１（アミノ酸配列：配列番号２）をコードする塩基配列（配列番号８）を含む発現ベクターｐＥＴ−ＦｃＲｎ＿ｍ１１、およびそれを含有する形質転換体である組換え大腸菌ＢＬ２１（ＤＥ３）／ＦｃＲｎ＿ｍ１１を取得した。
The absorbance at 450 nm of the ELISA method correlates with the amount of recombinant protein having antibody binding properties. Transformants with significantly increased absorbance were selected from random mutant libraries as compared to the culture supernatant of recombinant E. coli BL21 (DE3) / pET-FcRn_m7 (comparative control: producing the Fc-binding protein FcRn_m7). Expression vectors were extracted from each of the selected transformants, and the nucleotide sequence was analyzed.
(3) Fc-binding protein candidates As a result of the screening in (2) above, transformants capable of expressing the following Fc-binding proteins were obtained.
(3-1) Fc-binding protein FcRn_m8a
Expression vector pET-FcRn_m8a containing the nucleotide sequence (SEQ ID NO: 15) encoding the Fc-binding protein FcRn_m8a (amino acid sequence: SEQ ID NO: 14), and recombinant Escherichia coli BL21 (DE3) / FcRn_m8a containing it. Was acquired.
(3-2) Fc-binding protein FcRn_m8b
Expression vector pET-FcRn_m8b containing the nucleotide sequence (SEQ ID NO: 17) encoding the Fc-binding protein FcRn_m8b (amino acid sequence: SEQ ID NO: 16), and recombinant Escherichia coli BL21 (DE3) / FcRn_m8b containing it. Was obtained.
(3-3) Fc-binding protein FcRn_m11
Expression vector pET-FcRn_m11 containing a nucleotide sequence (SEQ ID NO: 8) encoding the Fc-binding protein FcRn_m11 (amino acid sequence: SEQ ID NO: 2), and recombinant Escherichia coli BL21 (DE3) / FcRn_m11 containing it. Was acquired.
参考例１ Ｆｃ結合性タンパク質ＦｃＲｎ＿ｍ８ａの製造と評価
実施例１の（３−１）に記載のＦｃ結合性タンパク質ＦｃＲｎ＿ｍ８ａのアミノ酸配列（配列番号１４）は、配列番号１のアミノ酸配列に対して前記の置換（Ｅ）（配列番号１の３０６番目のアスパラギンがアスパラギン酸に置換）が生じたアミノ酸配列である。
（１）Ｆｃ結合性タンパク質ＦｃＲｎ＿ｍ８ａの製造
製造は、実施例１の（３−１）に記載の組換え大腸菌ＢＬ２１（ＤＥ３）／ＦｃＲｎ＿ｍ８ａを使用して比較例１の（１−２）と同様の方法で行った。
（２）Ｆｃ結合性タンパク質ＦｃＲｎ＿ｍ８ａの生産性評価
生産性評価は、実施例１の（３−１）に記載の組換え大腸菌ＢＬ２１（ＤＥ３）／ＦｃＲｎ＿ｍ８ａを使用して比較例１の（２）と同様の方法で行った。比較例１の（１）で作製した精製済みのＦｃ結合性タンパク質ＦｃＲｎ＿ｍ７を濃度の標準として使用した。その結果、Ｆｃ結合性タンパク質ＦｃＲｎ＿ｍ８ａの発現量は５０ｍｇ／Ｌであった。
（３）Ｆｃ結合性タンパク質ＦｃＲｎ＿ｍ８ａの抗体に対する結合親和性評価
抗体に対する結合親和性評価は、精製済みのＦｃ結合性タンパク質ＦｃＲｎ＿ｍ８ａを用いて、比較例１の（３）と同様の方法で行った。その結果、Ｆｃ結合性タンパク質ＦｃＲｎ＿ｍ８ａのヒト抗体に対する解離定数（ＫＤ）の値は１７±４μＭ（ｎ＝４）であった。この値は、比較例１に記載のＦｃ結合性タンパク質ＦｃＲｎ＿ｍ７のヒト抗体に対する解離定数（ＫＤ）の値（１０±４μＭ）に近く、抗体に対する結合親和性が維持されていることが判明した。
Reference Example 1 Production and evaluation of the Fc-binding protein FcRn_m8a The amino acid sequence (SEQ ID NO: 14) of the Fc-binding protein FcRn_m8a according to (3-1) of Example 1 is the same as that of the amino acid sequence of SEQ ID NO: 1. It is an amino acid sequence in which substitution (E) (aspartic acid at position 306 of SEQ ID NO: 1 is replaced with aspartic acid) has occurred.
(1) Production of Fc-binding protein FcRn_m8a The production of the FcRn_m8a is the same as that of Comparative Example 1 (1-2) using the recombinant Escherichia coli BL21 (DE3) / FcRn_m8a described in (3-1) of Example 1. Got the way.
(2) Productivity evaluation of Fc-binding protein FcRn_m8a Productivity evaluation was carried out using the recombinant Escherichia coli BL21 (DE3) / FcRn_m8a described in (3-1) of Example 1 with (2) of Comparative Example 1. It was done in the same way. The purified Fc-binding protein FcRn_m7 prepared in Comparative Example 1 (1) was used as a concentration standard. As a result, the expression level of the Fc-binding protein FcRn_m8a was 50 mg / L.
(3) Evaluation of binding affinity of Fc-binding protein FcRn_m8a for antibody The evaluation of binding affinity for antibody was performed using the purified Fc-binding protein FcRn_m8a in the same manner as in (3) of Comparative Example 1. As a result, the value of the dissociation constant for human antibody Fc binding protein FcRn_m8a (K D) was 17 ± 4μM (n = 4) . This value was found to close to the value (10 ± 4 [mu] M) dissociation constant for human antibody Fc binding protein FcRn_m7 (K D), binding affinity for the antibody is maintained according to Comparative Example 1.
参考例２ Ｆｃ結合性タンパク質ＦｃＲｎ＿ｍ８ｂの製造と評価
実施例１の（３−２）に記載のＦｃ結合性タンパク質ＦｃＲｎ＿ｍ８ｂのアミノ酸配列（配列番号１６）は、配列番号１のアミノ酸配列に対して前記の置換（Ｆ）（配列番号１の３１５番目のセリンがスレオニンに置換）が生じたアミノ酸配列である。
（１）Ｆｃ結合性タンパク質ＦｃＲｎ＿ｍ８ｂの製造
製造は、実施例１の（３−２）に記載の組換え大腸菌ＢＬ２１（ＤＥ３）／ＦｃＲｎ＿ｍ８ｂを使用して比較例１の（１−２）と同様の方法で行った。
（２）Ｆｃ結合性タンパク質ＦｃＲｎ＿ｍ８ｂの生産性評価
生産性評価は、実施例１の（３−２）に記載の組換え大腸菌ＢＬ２１（ＤＥ３）／ＦｃＲｎ＿ｍ８ｂを使用して比較例１の（２）と同様の方法で行った。比較例１の（１）で作製した精製済みのＦｃ結合性タンパク質ＦｃＲｎ＿ｍ７を濃度の標準として使用した。その結果、Ｆｃ結合性タンパク質ＦｃＲｎ＿ｍ８ｂの発現量は３８ｍｇ／Ｌであった。
（３）Ｆｃ結合性タンパク質ＦｃＲｎ＿ｍ８ｂの抗体に対する結合親和性評価
抗体に対する結合親和性評価は、精製済みのＦｃ結合性タンパク質ＦｃＲｎ＿ｍ８ｂを用いて、比較例１の（３）と同様の方法で行った。その結果、Ｆｃ結合性タンパク質ＦｃＲｎ＿ｍ８ｂのヒト抗体に対する解離定数（ＫＤ）の値は１６±６μＭ（ｎ＝４）であった。この値は、比較例１に記載のＦｃ結合性タンパク質ＦｃＲｎ＿ｍ７のヒト抗体に対する解離定数（ＫＤ）の値（１０±４μＭ）に近く、抗体に対する結合親和性が維持されていることが判明した。
Reference Example 2 Production and Evaluation of Fc-Binding Protein FcRn_m8b The amino acid sequence (SEQ ID NO: 16) of the Fc-binding protein FcRn_m8b according to (3-2) of Example 1 is the same as that of the amino acid sequence of SEQ ID NO: 1. This is an amino acid sequence in which substitution (F) (serine at position 315 of SEQ ID NO: 1 is replaced with threonine) has occurred.
(1) Production of Fc-binding protein FcRn_m8b The production of the FcRn_m8b is the same as that of Comparative Example 1 (1-2) using the recombinant Escherichia coli BL21 (DE3) / FcRn_m8b described in (3-2) of Example 1. Got the way.
(2) Productivity evaluation of Fc-binding protein FcRn_m8b Productivity evaluation was carried out using the recombinant Escherichia coli BL21 (DE3) / FcRn_m8b described in (3-2) of Example 1 with (2) of Comparative Example 1. It was done in the same way. The purified Fc-binding protein FcRn_m7 prepared in Comparative Example 1 (1) was used as a concentration standard. As a result, the expression level of the Fc-binding protein FcRn_m8b was 38 mg / L.
(3) Evaluation of binding affinity of Fc-binding protein FcRn_m8b for antibody The evaluation of binding affinity for antibody was performed using the purified Fc-binding protein FcRn_m8b in the same manner as in (3) of Comparative Example 1. As a result, the value of the dissociation constant for human antibody Fc binding protein FcRn_m8b (K D) was 16 ± 6μM (n = 4) . This value was found to close to the value (10 ± 4 [mu] M) dissociation constant for human antibody Fc binding protein FcRn_m7 (K D), binding affinity for the antibody is maintained according to Comparative Example 1.
実施例２ Ｆｃ結合性タンパク質ＦｃＲｎ＿ｍ１１の製造と評価
実施例１の（３−３）に記載のＦｃ結合性タンパク質ＦｃＲｎ＿ｍ１１のアミノ酸配列（配列番号２）は、配列番号１のアミノ酸配列に対して前記の置換（Ａ）（配列番号１の２１４番目のバリンがアスパラギン酸に置換）、置換（Ｂ）（配列番号１の２３０番目のリジンがグルタミン酸に置換）、置換（Ｃ）（配列番号１の２４２番目のリジンがグルタミン酸に置換）および置換（Ｄ）（配列番号１の４００番目のリジンがグルタミン酸に置換）が生じたアミノ酸配列である。
（１）Ｆｃ結合性タンパク質ＦｃＲｎ＿ｍ１１の製造
製造は、実施例１の（３−３）に記載の組換え大腸菌ＢＬ２１（ＤＥ３）／ＦｃＲｎ＿ｍ１１を使用して比較例１の（１−２）と同様の方法で行った。
（２）Ｆｃ結合性タンパク質ＦｃＲｎ＿ｍ１１の生産性評価
生産性評価は、実施例１の（３−３）に記載の組換え大腸菌ＢＬ２１（ＤＥ３）／ＦｃＲｎ＿ｍ１１を使用して比較例１の（２）と同様の方法で行った。比較例１の（１）で作製した精製済みのＦｃ結合性タンパク質ＦｃＲｎ＿ｍ７を濃度の標準として使用した。その結果、Ｆｃ結合性タンパク質ＦｃＲｎ＿ｍ１１の発現量は６９ｍｇ／Ｌであり、比較例１に記載のＦｃ結合性タンパク質ＦｃＲｎ＿ｍ７の発現量（４５ｍｇ／Ｌ）より高く、前記の置換（Ａ）、置換（Ｂ）、置換（Ｃ）および置換（Ｄ）を有することで生産性が向上した（表１）。
（３）Ｆｃ結合性タンパク質ＦｃＲｎ＿ｍ１１の抗体に対する結合親和性評価
抗体に対する結合親和性評価は、精製済みのＦｃ結合性タンパク質ＦｃＲｎ＿ｍ１１を用いて、比較例１の（３）と同様の方法で行った。その結果、Ｆｃ結合性タンパク質ＦｃＲｎ＿ｍ１１のヒト抗体に対する解離定数（ＫＤ）の値は８±３μＭ（ｎ＝３）であった。この値は、比較例１に記載のＦｃ結合性タンパク質ＦｃＲｎ＿ｍ７のヒト抗体に対する解離定数（ＫＤ）の値（１０±４μＭ）に近く、抗体に対する結合親和性が維持されていることが判明した。
実施例３ Ｆｃ結合性タンパク質ＦｃＲｎ＿ｍ１２ａの製造と評価
Ｆｃ結合性タンパク質ＦｃＲｎ＿ｍ１２ａのアミノ酸配列を配列番号３に示す。なお、配列番号３において、その１番目のメチオニンから４３１番目のセリンまでは、配列番号１の１番目のメチオニンから４３１番目のセリンに相当するアミノ酸配列に対して前記の置換（Ａ）（配列番号１の２１４番目のバリンがアスパラギン酸に置換）、置換（Ｂ）（配列番号１の２３０番目のリジンがグルタミン酸に置換）、置換（Ｃ）（配列番号１の２４２番目のリジンがグルタミン酸に置換）および置換（Ｄ）（配列番号１の４００番目のリジンがグルタミン酸に置換）および置換（Ｅ）（配列番号１の３０６番目のアスパラギンがアスパラギン酸に置換）が生じたアミノ酸配列に相当する。配列番号３において、その４３２番目のロイシンから４３９番目のヒスチジンまではポリヒスチジン配列を含むオリゴペプチドである。
（１）Ｆｃ結合性タンパク質ＦｃＲｎ＿ｍ１２ａの製造
Ｆｃ結合性タンパク質ＦｃＲｎ＿ｍ１２ａをコードする塩基配列（配列番号９）を含む発現ベクターｐＥＴ−ＦｃＲｎ＿ｍ１２ａおよびそれを有する形質転換体の組換え大腸菌ＢＬ２１（ＤＥ３）／ｐＥＴ−ＦｃＲｎ＿ｍ１２ａの作製は、下記の方法で行った。
Example 2 Production and evaluation of the Fc-binding protein FcRn_m11 The amino acid sequence (SEQ ID NO: 2) of the Fc-binding protein FcRn_m11 according to (3-3) of Example 1 is the above-mentioned with respect to the amino acid sequence of SEQ ID NO: 1. Substitution (A) (Valin at position 214 of SEQ ID NO: 1 is replaced with aspartic acid), Substitution (B) (Lysine at position 230 of SEQ ID NO: 1 is replaced with glutamic acid), Substitution (C) (Position 242 of SEQ ID NO: 1) Lysine is replaced with glutamic acid) and substitution (D) (the 400th lysine of SEQ ID NO: 1 is replaced with glutamic acid).
(1) Production of Fc-binding protein FcRn_m11 The production of the FcRn_m11 is the same as that of Comparative Example 1 (1-2) using the recombinant Escherichia coli BL21 (DE3) / FcRn_m11 described in (3-3) of Example 1. Got the way.
(2) Productivity evaluation of Fc-binding protein FcRn_m11 Productivity evaluation was carried out using the recombinant Escherichia coli BL21 (DE3) / FcRn_m11 described in (3-3) of Example 1 with (2) of Comparative Example 1. It was done in the same way. The purified Fc-binding protein FcRn_m7 prepared in Comparative Example 1 (1) was used as a concentration standard. As a result, the expression level of the Fc-binding protein FcRn_m11 was 69 mg / L, which was higher than the expression level of the Fc-binding protein FcRn_m7 described in Comparative Example 1 (45 mg / L), and the substitutions (A) and substitutions (B) were described above. ), Substitution (C) and Substitution (D) improved productivity (Table 1).
(3) Evaluation of binding affinity of Fc-binding protein FcRn_m11 for antibody The evaluation of binding affinity for antibody was performed using the purified Fc-binding protein FcRn_m11 in the same manner as in (3) of Comparative Example 1. As a result, the value of the dissociation constant for human antibody Fc binding protein FcRn_m11 (K D) was 8 ± 3μM (n = 3) . This value was found to close to the value (10 ± 4 [mu] M) dissociation constant for human antibody Fc binding protein FcRn_m7 (K D), binding affinity for the antibody is maintained according to Comparative Example 1.
Example 3 Production and Evaluation of Fc-Binding Protein FcRn_m12a The amino acid sequence of Fc-binding protein FcRn_m12a is shown in SEQ ID NO: 3. In SEQ ID NO: 3, the first methionine to the 431st serine of SEQ ID NO: 3 are substituted (A) (SEQ ID NO:) with respect to the amino acid sequence corresponding to the first methionine to the 431st serine of SEQ ID NO: 1. 1 214th valine is replaced with aspartic acid), substitution (B) (230th lysine of SEQ ID NO: 1 is replaced with glutamic acid), substitution (C) (242nd lysine of SEQ ID NO: 1 is replaced with glutamic acid) And Substitution (D) (400th lysine of SEQ ID NO: 1 is replaced with glutamic acid) and Substitution (E) (306th aspartic acid of SEQ ID NO: 1 is replaced with aspartic acid) corresponds to the amino acid sequence. In SEQ ID NO: 3, leucine at position 432 to histidine at position 439 are oligopeptides containing a polyhistidine sequence.
(1) Production of Fc-Binding Protein FcRn_m12a An expression vector pET-FcRn_m12a containing a nucleotide sequence (SEQ ID NO: 9) encoding the Fc-binding protein FcRn_m12a and a recombinant Escherichia coli BL21 (DE3) / pET- FcRn_m12a was prepared by the following method.
実施例１の（３−３）に記載の発現ベクターｐＥＴ−ＦｃＲｎ＿ｍ１１を鋳型として、配列番号１２および配列番号１８に記載の配列からなる各オリゴヌクレオチドをＰＣＲプライマーとして、比較例１の（１−１）と同様の方法によりＰＣＲを実施し、得られたＰＣＲ産物を制限酵素ＮｈｅＩおよびＳａｃＩで消化した。さらに、実施例１の（３−３）に記載の発現ベクターｐＥＴ−ＦｃＲｎ＿ｍ１１を鋳型として、配列番号１９および配列番号２０に記載の配列からなる各オリゴヌクレオチドをＰＣＲプライマーとして、同様にＰＣＲを実施し、得られたＰＣＲ産物を制限酵素ＳａｃＩおよびＸｈｏＩで消化した。制限酵素ＮｈｅＩおよびＸｈｏＩで制限酵素処理した比較例１の（１−１）に記載の発現ベクターｐＥＴ−ＦｃＲｎ＿ｍ７とライゲーション反応を行った。このライゲーション産物を用いて大腸菌ＢＬ２１（ＤＥ３）を形質転換し、組換え大腸菌ＢＬ２１（ＤＥ３）／ｐＥＴ−ＦｃＲｎ＿ｍ１２ａを得た。配列解析により塩基配列を確認した結果、発現ベクターｐＥＴ−ＦｃＲｎ＿ｍ１２ａには配列番号３のアミノ酸配列をコードする配列番号９の塩基配列が含まれることを確認した。 Using the expression vector pET-FcRn_m11 described in (3-3) of Example 1 as a template and each oligonucleotide consisting of the sequences shown in SEQ ID NO: 12 and SEQ ID NO: 18 as a PCR primer, (1-1) of Comparative Example 1 ) Was carried out, and the obtained PCR product was digested with restriction enzymes NheI and SacI. Further, PCR was carried out in the same manner using the expression vector pET-FcRn_m11 described in (3-3) of Example 1 as a template and each oligonucleotide consisting of the sequences shown in SEQ ID NO: 19 and SEQ ID NO: 20 as a PCR primer. , The obtained PCR product was digested with restriction enzymes SacI and XhoI. A ligation reaction was carried out with the expression vector pET-FcRn_m7 described in (1-1) of Comparative Example 1 treated with restriction enzymes NheI and XhoI. Escherichia coli BL21 (DE3) was transformed with this ligation product to obtain recombinant Escherichia coli BL21 (DE3) / pET-FcRn_m12a. As a result of confirming the base sequence by sequence analysis, it was confirmed that the expression vector pET-FcRn_m12a contains the base sequence of SEQ ID NO: 9 encoding the amino acid sequence of SEQ ID NO: 3.
Ｆｃ結合性タンパク質ＦｃＲｎ＿ｍ１２ａの製造は、前記の組換え大腸菌ＢＬ２１（ＤＥ３）／ｐＥＴ−ＦｃＲｎ＿ｍ１２ａを使用して比較例１の（１−２）と同様の方法で行った。
（２）Ｆｃ結合性タンパク質ＦｃＲｎ＿ｍ１２ａの生産性評価
生産性評価は、前記の組換え大腸菌ＢＬ２１（ＤＥ３）／ｐＥＴ−ＦｃＲｎ＿ｍ１２ａを使用して比較例１の（２）と同様の方法で行った。比較例１の（１）で作製した精製済みのＦｃ結合性タンパク質ＦｃＲｎ＿ｍ７を濃度の標準として使用した。その結果、Ｆｃ結合性タンパク質ＦｃＲｎ＿ｍ１２ａの発現量は１２５ｍｇ／Ｌであり、比較例１に記載のＦｃ結合性タンパク質ＦｃＲｎ＿ｍ７の発現量（４５ｍｇ／Ｌ）より高く、前記の置換（Ａ）、置換（Ｂ）、置換（Ｃ）、置換（Ｄ）および置換（Ｅ）を有することで生産性が向上した（表１）。
（３）Ｆｃ結合性タンパク質ＦｃＲｎ＿ｍ１２ａの抗体に対する結合親和性評価
抗体に対する結合親和性評価は、精製済みのＦｃ結合性タンパク質ＦｃＲｎ＿ｍ１２ａを用いて、比較例１の（３）と同様の方法で行った。その結果、Ｆｃ結合性タンパク質ＦｃＲｎ＿ｍ１２ａのヒト抗体に対する解離定数（ＫＤ）の値は１８±１１μＭ（ｎ＝３）であった。この値は、比較例１に記載のＦｃ結合性タンパク質ＦｃＲｎ＿ｍ７のヒト抗体に対する解離定数（ＫＤ）の値（１０±４μＭ）に近く、抗体に対する結合親和性が維持されていることが判明した。
実施例４ Ｆｃ結合性タンパク質ＦｃＲｎ＿ｍ１２ｂの製造と評価
Ｆｃ結合性タンパク質ＦｃＲｎ＿ｍ１２ｂのアミノ酸配列を配列番号４に示す。なお、配列番号４において、その１番目のメチオニンから４３１番目のセリンまでは、配列番号１の１番目のメチオニンから４３１番目のセリンに相当するアミノ酸配列に対して前記の置換（Ａ）（配列番号１の２１４番目のバリンがアスパラギン酸に置換）、置換（Ｂ）（配列番号１の２３０番目のリジンがグルタミン酸に置換）、置換（Ｃ）（配列番号１の２４２番目のリジンがグルタミン酸に置換）および置換（Ｄ）（配列番号１の４００番目のリジンがグルタミン酸に置換）および置換（Ｆ）（配列番号１の３１５番目のセリンがスレオニンに置換）が生じたアミノ酸配列に相当する。配列番号４において、その４３２番目のロイシンから４３９番目のヒスチジンまではポリヒスチジン配列を含むオリゴペプチドである。
（１）Ｆｃ結合性タンパク質ＦｃＲｎ＿ｍ１２ｂの製造
Ｆｃ結合性タンパク質ＦｃＲｎ＿ｍ１２ｂをコードする塩基配列（配列番号１０）を含む発現ベクターｐＥＴ−ＦｃＲｎ＿ｍ１２ｂおよびそれを有する形質転換体の組換え大腸菌ＢＬ２１（ＤＥ３）／ｐＥＴ−ＦｃＲｎ＿ｍ１２ｂの作製は、下記の方法で行った。
The Fc-binding protein FcRn_m12a was produced using the above-mentioned recombinant Escherichia coli BL21 (DE3) / pET-FcRn_m12a in the same manner as in Comparative Example 1 (1-2).
(2) Productivity Evaluation of Fc-Binding Protein FcRn_m12a Productivity evaluation was carried out in the same manner as in (2) of Comparative Example 1 using the above-mentioned recombinant Escherichia coli BL21 (DE3) / pET-FcRn_m12a. The purified Fc-binding protein FcRn_m7 prepared in Comparative Example 1 (1) was used as a concentration standard. As a result, the expression level of the Fc-binding protein FcRn_m12a was 125 mg / L, which was higher than the expression level of the Fc-binding protein FcRn_m7 described in Comparative Example 1 (45 mg / L). ), Substitution (C), Substitution (D) and Substitution (E) improved productivity (Table 1).
(3) Evaluation of binding affinity of Fc-binding protein FcRn_m12a for antibody The evaluation of binding affinity for antibody was performed using the purified Fc-binding protein FcRn_m12a in the same manner as in (3) of Comparative Example 1. As a result, the value of the dissociation constant for human antibody Fc binding protein FcRn_m12a (K D) was 18 ± 11μM (n = 3) . This value was found to close to the value (10 ± 4 [mu] M) dissociation constant for human antibody Fc binding protein FcRn_m7 (K D), binding affinity for the antibody is maintained according to Comparative Example 1.
Example 4 Production and Evaluation of Fc-Binding Protein FcRn_m12b The amino acid sequence of Fc-binding protein FcRn_m12b is shown in SEQ ID NO: 4. In SEQ ID NO: 4, the first methionine to the 431st serine of SEQ ID NO: 1 are substituted (A) (SEQ ID NO:) with respect to the amino acid sequence corresponding to the first methionine to the 431st serine of SEQ ID NO: 1. 1 214th valine is replaced with aspartic acid), substitution (B) (230th lysine of SEQ ID NO: 1 is replaced with glutamic acid), substitution (C) (242nd lysine of SEQ ID NO: 1 is replaced with glutamic acid) And Substitution (D) (the 400th lysine of SEQ ID NO: 1 is replaced with glutamic acid) and Substitution (F) (the 315th serine of SEQ ID NO: 1 is replaced with methionine) corresponds to the amino acid sequence. In SEQ ID NO: 4, leucine at position 432 to histidine at position 439 are oligopeptides containing a polyhistidine sequence.
(1) Production of Fc-Binding Protein FcRn_m12b An expression vector pET-FcRn_m12b containing a nucleotide sequence (SEQ ID NO: 10) encoding the Fc-binding protein FcRn_m12b and a recombinant Escherichia coli BL21 (DE3) / pET- FcRn_m12b was prepared by the following method.
実施例１の（３−３）に記載の発現ベクターｐＥＴ−ＦｃＲｎ＿ｍ１１を鋳型として、配列番号１２および配列番号２１に記載の配列からなる各オリゴヌクレオチドをＰＣＲプライマーとして、比較例１の（１−１）と同様の方法によりＰＣＲを実施し、得られたＰＣＲ産物を制限酵素ＮｈｅＩおよびＳａｃＩで消化した。さらに、実施例１の（３−３）に記載の発現ベクターｐＥＴ−ＦｃＲｎ＿ｍ１１を鋳型として、配列番号２２および配列番号２０に記載の配列からなる各オリゴヌクレオチドをＰＣＲプライマーとして、同様にＰＣＲを実施し、得られたＰＣＲ産物を制限酵素ＳａｃＩおよびＸｈｏＩで消化した。これらの制限酵素消化産物を制限酵素ＮｈｅＩおよびＸｈｏＩで制限酵素処理した比較例１の（１−１）に記載の発現ベクターｐＥＴ−ＦｃＲｎ＿ｍ７とライゲーション反応を行った。このライゲーション産物を用いて大腸菌ＢＬ２１（ＤＥ３）を形質転換し、組換え大腸菌ＢＬ２１（ＤＥ３）／ｐＥＴ−ＦｃＲｎ＿ｍ１２ｂを得た。配列解析により塩基配列を確認した結果、発現ベクターｐＥＴ−ＦｃＲｎ＿ｍ１２ｂには配列番号４のアミノ酸配列をコードする配列番号１０の塩基配列が含まれることを確認した。 Using the expression vector pET-FcRn_m11 described in (3-3) of Example 1 as a template and each oligonucleotide consisting of the sequences shown in SEQ ID NO: 12 and SEQ ID NO: 21 as a PCR primer, (1-1) of Comparative Example 1 ) Was carried out, and the obtained PCR product was digested with restriction enzymes NheI and SacI. Further, PCR was carried out in the same manner using the expression vector pET-FcRn_m11 described in (3-3) of Example 1 as a template and each oligonucleotide consisting of the sequences shown in SEQ ID NO: 22 and SEQ ID NO: 20 as a PCR primer. , The obtained PCR product was digested with restriction enzymes SacI and XhoI. These restriction enzyme digested products were treated with restriction enzymes NheI and XhoI, and a ligation reaction was carried out with the expression vector pET-FcRn_m7 described in Comparative Example 1 (1-1). Escherichia coli BL21 (DE3) was transformed with this ligation product to obtain recombinant Escherichia coli BL21 (DE3) / pET-FcRn_m12b. As a result of confirming the base sequence by sequence analysis, it was confirmed that the expression vector pET-FcRn_m12b contains the base sequence of SEQ ID NO: 10 encoding the amino acid sequence of SEQ ID NO: 4.
Ｆｃ結合性タンパク質ＦｃＲｎ＿ｍ１２ｂの製造は、前記の組換え大腸菌ＢＬ２１（ＤＥ３）／ｐＥＴ−ＦｃＲｎ＿ｍ１２ｂを使用して比較例１の（１−２）と同様の方法で行った。
（２）Ｆｃ結合性タンパク質ＦｃＲｎ＿ｍ１２ｂの生産性評価
生産性評価は、前記の組換え大腸菌ＢＬ２１（ＤＥ３）／ｐＥＴ−ＦｃＲｎ＿ｍ１２ｂを使用して比較例１の（２）と同様の方法で行った。比較例１の（１）で作製した精製済みのＦｃ結合性タンパク質ＦｃＲｎ＿ｍ７を濃度の標準として使用した。その結果、Ｆｃ結合性タンパク質ＦｃＲｎ＿ｍ１２ｂの発現量は１５９ｍｇ／Ｌであり、比較例１に記載のＦｃ結合性タンパク質ＦｃＲｎ＿ｍ７の発現量（４５ｍｇ／Ｌ）より高く、前記の置換（Ａ）、置換（Ｂ）、置換（Ｃ）、置換（Ｄ）および置換（Ｆ）を有することで生産性が向上した（表１）。
（３）Ｆｃ結合性タンパク質ＦｃＲｎ＿ｍ１２ｂの抗体に対する結合親和性評価
抗体に対する結合親和性評価は、精製済みのＦｃ結合性タンパク質ＦｃＲｎ＿ｍ１２ｂを用いて、比較例１の（３）と同様の方法で行った。その結果、Ｆｃ結合性タンパク質ＦｃＲｎ＿ｍ１２ｂのヒト抗体に対する解離定数（ＫＤ）の値は１５±８μＭ（ｎ＝３）であった。この値は、比較例１に記載のＦｃ結合性タンパク質ＦｃＲｎ＿ｍ７のヒト抗体に対する解離定数（ＫＤ）の値（１０±４μＭ）に近く、抗体に対する結合親和性が維持されていることが判明した。
The Fc-binding protein FcRn_m12b was produced using the above-mentioned recombinant Escherichia coli BL21 (DE3) / pET-FcRn_m12b in the same manner as in Comparative Example 1 (1-2).
(2) Productivity Evaluation of Fc-Binding Protein FcRn_m12b Productivity evaluation was carried out in the same manner as in (2) of Comparative Example 1 using the above-mentioned recombinant Escherichia coli BL21 (DE3) / pET-FcRn_m12b. The purified Fc-binding protein FcRn_m7 prepared in Comparative Example 1 (1) was used as a concentration standard. As a result, the expression level of the Fc-binding protein FcRn_m12b was 159 mg / L, which was higher than the expression level of the Fc-binding protein FcRn_m7 described in Comparative Example 1 (45 mg / L). ), Substitution (C), Substitution (D) and Substitution (F) improved productivity (Table 1).
(3) Evaluation of binding affinity of Fc-binding protein FcRn_m12b for antibody The evaluation of binding affinity for antibody was performed using the purified Fc-binding protein FcRn_m12b in the same manner as in (3) of Comparative Example 1. As a result, the value of the dissociation constant for human antibody Fc binding protein FcRn_m12b (K D) was 15 ± 8μM (n = 3) . This value was found to close to the value (10 ± 4 [mu] M) dissociation constant for human antibody Fc binding protein FcRn_m7 (K D), binding affinity for the antibody is maintained according to Comparative Example 1.
実施例５ Ｆｃ結合性タンパク質ＦｃＲｎ＿ｍ１３の製造と評価
Ｆｃ結合性タンパク質ＦｃＲｎ＿ｍ１３のアミノ酸配列を配列番号５に示す。なお、配列番号５において、その１番目のメチオニンから４３１番目のセリンまでは、配列番号１の１番目のメチオニンから４３１番目のセリンに相当するアミノ酸配列に対して前記の置換（Ａ）（配列番号１の２１４番目のバリンがアスパラギン酸に置換）、置換（Ｂ）（配列番号１の２３０番目のリジンがグルタミン酸に置換）、置換（Ｃ）（配列番号１の２４２番目のリジンがグルタミン酸に置換）および置換（Ｄ）（配列番号１の４００番目のリジンがグルタミン酸に置換）、置換（Ｅ）（配列番号１の３０６番目のアスパラギンがアスパラギン酸に置換））および置換（Ｆ）（配列番号１の３１５番目のセリンがスレオニンに置換）が生じたアミノ酸配列に相当する。配列番号５において、その４３２番目のロイシンから４３９番目のヒスチジンまではポリヒスチジン配列を含むオリゴペプチドである。
（１）Ｆｃ結合性タンパク質ＦｃＲｎ＿ｍ１３の製造
Ｆｃ結合性タンパク質ＦｃＲｎ＿ｍ１３をコードする塩基配列（配列番号１１）を含む発現ベクターｐＥＴ−ＦｃＲｎ＿ｍ１３およびそれを有する形質転換体の組換え大腸菌ＢＬ２１（ＤＥ３）／ｐＥＴ−ＦｃＲｎ＿ｍ１３の作製は、下記の方法で行った。
Example 5 Production and Evaluation of Fc-Binding Protein FcRn_m13 The amino acid sequence of Fc-binding protein FcRn_m13 is shown in SEQ ID NO: 5. In SEQ ID NO: 5, the first methionine to the 431st serine of SEQ ID NO: 1 are substituted (A) (SEQ ID NO:) with respect to the amino acid sequence corresponding to the first methionine to the 431st serine of SEQ ID NO: 1. 1 214th valine is replaced with aspartic acid), substitution (B) (230th lysine of SEQ ID NO: 1 is replaced with glutamic acid), substitution (C) (242nd lysine of SEQ ID NO: 1 is replaced with glutamic acid) And Substitution (D) (400th lysine of SEQ ID NO: 1 is replaced with glutamic acid), Substitution (E) (306th aspartic acid of SEQ ID NO: 1 is replaced with aspartic acid)) and Substitution (F) (of SEQ ID NO: 1 Corresponds to the amino acid sequence in which serine at position 315 was replaced with threonine). In SEQ ID NO: 5, leucine at position 432 to histidine at position 439 are oligopeptides containing a polyhistidine sequence.
(1) Production of Fc-binding protein FcRn_m13 An expression vector pET-FcRn_m13 containing a nucleotide sequence encoding Fc-binding protein FcRn_m13 (SEQ ID NO: 11) and a recombinant Escherichia coli BL21 (DE3) / pET- of a transformant having the same. FcRn_m13 was prepared by the following method.
実施例１の（３−３）に記載の発現ベクターｐＥＴ−ＦｃＲｎ＿ｍ１１を鋳型として、配列番号１２および配列番号１８に記載の配列からなる各オリゴヌクレオチドをＰＣＲプライマーとして、比較例１の（１−１）と同様の方法によりＰＣＲを実施し、得られたＰＣＲ産物を制限酵素ＮｈｅＩおよびＳａｃＩで消化した。さらに、実施例１の（３−３）に記載の発現ベクターｐＥＴ−ＦｃＲｎ＿ｍ１１を鋳型として、配列番号２２および配列番号２０に記載の配列からなる各オリゴヌクレオチドをＰＣＲプライマーとして、同様にＰＣＲを実施し、得られたＰＣＲ産物を制限酵素ＳａｃＩおよびＸｈｏＩで消化した。これらの制限酵素消化産物を制限酵素ＮｈｅＩおよびＸｈｏＩで制限酵素処理した比較例１の（１−１）に記載の発現ベクターｐＥＴ−ＦｃＲｎ＿ｍ７とライゲーション反応を行った。このライゲーション産物を用いて大腸菌ＢＬ２１（ＤＥ３）を形質転換し、組換え大腸菌ＢＬ２１（ＤＥ３）／ｐＥＴ−ＦｃＲｎ＿ｍ１３を得た。配列解析により塩基配列を確認した結果、発現ベクターｐＥＴ−ＦｃＲｎ＿ｍ１３には配列番号５のアミノ酸配列をコードする配列番号１１の塩基配列が含まれることを確認した。 Using the expression vector pET-FcRn_m11 described in (3-3) of Example 1 as a template and each oligonucleotide consisting of the sequences shown in SEQ ID NO: 12 and SEQ ID NO: 18 as a PCR primer, (1-1) of Comparative Example 1 ) Was carried out, and the obtained PCR product was digested with restriction enzymes NheI and SacI. Further, PCR was carried out in the same manner using the expression vector pET-FcRn_m11 described in (3-3) of Example 1 as a template and each oligonucleotide consisting of the sequences shown in SEQ ID NO: 22 and SEQ ID NO: 20 as a PCR primer. , The obtained PCR product was digested with restriction enzymes SacI and XhoI. These restriction enzyme digested products were treated with restriction enzymes NheI and XhoI, and a ligation reaction was carried out with the expression vector pET-FcRn_m7 described in Comparative Example 1 (1-1). Escherichia coli BL21 (DE3) was transformed with this ligation product to obtain recombinant Escherichia coli BL21 (DE3) / pET-FcRn_m13. As a result of confirming the base sequence by sequence analysis, it was confirmed that the expression vector pET-FcRn_m13 contains the base sequence of SEQ ID NO: 11 encoding the amino acid sequence of SEQ ID NO: 5.
Ｆｃ結合性タンパク質ＦｃＲｎ＿ｍ１３の製造は、前記の組換え大腸菌ＢＬ２１（ＤＥ３）／ｐＥＴ−ＦｃＲｎ＿ｍ１３を使用して比較例１の（１−２）と同様の方法で行った。
（２）Ｆｃ結合性タンパク質ＦｃＲｎ＿ｍ１３の生産性評価
生産性評価は、前記の組換え大腸菌ＢＬ２１（ＤＥ３）／ｐＥＴ−ＦｃＲｎ＿ｍ１３を使用して比較例１の（２）と同様の方法で行った。比較例１の（１）で作製した精製済みのＦｃ結合性タンパク質ＦｃＲｎ＿ｍ７を濃度の標準として使用した。その結果、Ｆｃ結合性タンパク質ＦｃＲｎ＿ｍ１３の発現量は１５６ｍｇ／Ｌであり、比較例１に記載のＦｃ結合性タンパク質ＦｃＲｎ＿ｍ７の発現量（４５ｍｇ／Ｌ）より高く、前記の置換（Ａ）、置換（Ｂ）、置換（Ｃ）、置換（Ｄ）、置換（Ｅ）および置換（Ｆ）を有することで生産性が向上した（表１）。
（３）Ｆｃ結合性タンパク質ＦｃＲｎ＿ｍ１３の抗体に対する結合親和性評価
抗体に対する結合親和性評価は、精製済みのＦｃ結合性タンパク質ＦｃＲｎ＿ｍ１３を用いて、比較例１の（３）と同様の方法で行った。その結果、Ｆｃ結合性タンパク質ＦｃＲｎ＿ｍ１３のヒト抗体に対する解離定数（ＫＤ）の値は１２±１μＭ（ｎ＝３）であった。この値は、比較例１に記載のＦｃ結合性タンパク質ＦｃＲｎ＿ｍ７のヒト抗体に対する解離定数（ＫＤ）の値（１０±４μＭ）に近く、抗体に対する結合親和性が維持されていることが判明した。
（４）Ｆｃ結合性タンパク質ＦｃＲｎ＿ｍ１３の熱安定性評価
精製済みのＦｃ結合性タンパク質ＦｃＲｎ＿ｍ１３を用いて、比較例１の（４）と同様の方法で熱安定性評価を行った。
The Fc-binding protein FcRn_m13 was produced using the above-mentioned recombinant Escherichia coli BL21 (DE3) / pET-FcRn_m13 in the same manner as in Comparative Example 1 (1-2).
(2) Productivity Evaluation of Fc-Binding Protein FcRn_m13 Productivity evaluation was carried out in the same manner as in (2) of Comparative Example 1 using the above-mentioned recombinant Escherichia coli BL21 (DE3) / pET-FcRn_m13. The purified Fc-binding protein FcRn_m7 prepared in Comparative Example 1 (1) was used as a concentration standard. As a result, the expression level of the Fc-binding protein FcRn_m13 was 156 mg / L, which was higher than the expression level of the Fc-binding protein FcRn_m7 described in Comparative Example 1 (45 mg / L). ), Substitution (C), Substitution (D), Substitution (E) and Substitution (F) improved productivity (Table 1).
(3) Evaluation of binding affinity of Fc-binding protein FcRn_m13 for antibody The evaluation of binding affinity for antibody was performed using the purified Fc-binding protein FcRn_m13 in the same manner as in (3) of Comparative Example 1. As a result, the value of the dissociation constant for human antibody Fc binding protein FcRn_m13 (K D) was 12 ± 1μM (n = 3) . This value was found to close to the value (10 ± 4 [mu] M) dissociation constant for human antibody Fc binding protein FcRn_m7 (K D), binding affinity for the antibody is maintained according to Comparative Example 1.
(4) Thermal stability evaluation of Fc-binding protein FcRn_m13 Using the purified Fc-binding protein FcRn_m13, the thermal stability was evaluated in the same manner as in (4) of Comparative Example 1.
Ｆｃ結合性タンパク質ＦｃＲｎ＿ｍ１３の変性中点は６４±２℃（ｎ＝２）であり、比較例１の（４）に記載のＦｃ結合性タンパク質ＦｃＲｎ＿ｍ７の変性中点（６０±２℃）より高く、前記の置換（Ａ）、置換（Ｂ）、置換（Ｃ）、置換（Ｄ）、置換（Ｅ）および置換（Ｆ）を有することで熱安定性が向上したことが判明した。 The mid-denaturation point of the Fc-binding protein FcRn_m13 is 64 ± 2 ° C. (n = 2), which is higher than the mid-denaturation point (60 ± 2 ° C.) of the Fc-binding protein FcRn_m7 described in (4) of Comparative Example 1. It was found that the thermal stability was improved by having the substitutions (A), substitutions (B), substitutions (C), substitutions (D), substitutions (E) and substitutions (F).
本発明のＦｃ結合性タンパク質は、ＩｇＧまたはＦｃ融合タンパク質を分離するための吸着剤のリガンドとして有用である。 The Fc-binding proteins of the present invention are useful as ligands for adsorbents for separating IgG or Fc fusion proteins.
Claims (11)
（Ａ）配列番号１の２１４番目のバリンがアスパラギン酸に置換、
（Ｂ）配列番号１の２３０番目のリジンがグルタミン酸に置換、
（Ｃ）配列番号１の２４２番目のリジンがグルタミン酸に置換、
（Ｄ）配列番号１の４００番目のリジンがグルタミン酸に置換、
（Ｅ）配列番号１の３０６番目のアスパラギンがアスパラギン酸に置換、
（Ｆ）配列番号１の３１５番目のセリンがスレオニンに置換。 An Fc-binding protein comprising an amino acid sequence having at least one of the following amino acid substitutions (A) to (F) in the amino acid residues 34 to 431 of the amino acid sequence set forth in SEQ ID NO: 1.
(A) Valine at position 214 of SEQ ID NO: 1 is replaced with aspartic acid,
(B) The 230th lysine of SEQ ID NO: 1 is replaced with glutamic acid,
(C) The 242nd lysine of SEQ ID NO: 1 is replaced with glutamic acid,
(D) The 400th lysine of SEQ ID NO: 1 is replaced with glutamic acid,
(E) Asparagine at position 306 of SEQ ID NO: 1 is replaced with aspartic acid,
(F) Serine at position 315 of SEQ ID NO: 1 is replaced with threonine.
（１）配列番号１に記載のアミノ酸配列の３４番目から４３１番目までのアミノ酸残基において、前記１以上のアミノ酸置換を有するアミノ酸配列を含む、Ｆｃ結合性タンパク質；
（２）配列番号１に記載のアミノ酸配列の３４番目から４３１番目までのアミノ酸残基において、前記１以上のアミノ酸置換を有し、さらに前記少なくとも１つのアミノ酸置換の位置以外の１若しくは数個の位置での１若しくは数個のアミノ酸残基の置換、欠失、挿入および付加のうち、いずれか１つ以上を有するアミノ酸配列を含む、Ｆｃ結合性タンパク質；
（３）配列番号１に記載のアミノ酸配列の３４番目から４３１番目までのアミノ酸残基において、前記１以上のアミノ酸置換を有するアミノ酸配列全体に対して８０％以上の相同性を有するアミノ酸配列であって、前記１以上のアミノ酸置換が残存したアミノ酸配列を含む、Ｆｃ結合性タンパク質。 Fc-binding protein, which is the protein according to any one of the following (1) to (3):
(1) An Fc-binding protein containing an amino acid sequence having one or more amino acid substitutions at the 34th to 431st amino acid residues of the amino acid sequence shown in SEQ ID NO: 1.
(2) At the amino acid residues 34 to 431 of the amino acid sequence shown in SEQ ID NO: 1, one or several amino acid residues other than the position of at least one amino acid substitution having one or more amino acid substitutions. An Fc-binding protein comprising an amino acid sequence having one or more of substitutions, deletions, insertions and additions of one or several amino acid residues at a position;
(3) The amino acid residues from the 34th to the 431st of the amino acid sequence shown in SEQ ID NO: 1 are amino acid sequences having 80% or more homology with the entire amino acid sequence having the amino acid substitution of 1 or more. An Fc-binding protein comprising an amino acid sequence in which one or more amino acid substitutions remain.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2019202298A JP7435939B2 (en) | 2019-11-07 | 2019-11-07 | Highly productive Fc-binding protein and method for producing the same |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2019202298A JP7435939B2 (en) | 2019-11-07 | 2019-11-07 | Highly productive Fc-binding protein and method for producing the same |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JP2021073883A true JP2021073883A (en) | 2021-05-20 |
| JP7435939B2 JP7435939B2 (en) | 2024-02-21 |
Family
ID=75897045
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2019202298A Active JP7435939B2 (en) | 2019-11-07 | 2019-11-07 | Highly productive Fc-binding protein and method for producing the same |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP7435939B2 (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2011111393A1 (en) * | 2010-03-10 | 2011-09-15 | 公益財団法人相模中央化学研究所 | Fc binding protein and method for producing same |
| JP2017178908A (en) * | 2016-03-31 | 2017-10-05 | 東ソー株式会社 | MODIFICATION TYPE RECOMBINANT FcγRIIb |
| WO2018056374A1 (en) * | 2016-09-23 | 2018-03-29 | 東ソー株式会社 | IMPROVED RECOMBINANT FcγRII |
| WO2018198817A1 (en) * | 2017-04-26 | 2018-11-01 | 東ソー株式会社 | Stable fc binding protein, method for producing said protein, and antibody adsorbent in which said protein is used |
| JP2019076069A (en) * | 2017-10-27 | 2019-05-23 | 東ソー株式会社 | Fc-binding proteins with amino acid substitution |
-
2019
- 2019-11-07 JP JP2019202298A patent/JP7435939B2/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2011111393A1 (en) * | 2010-03-10 | 2011-09-15 | 公益財団法人相模中央化学研究所 | Fc binding protein and method for producing same |
| JP2017178908A (en) * | 2016-03-31 | 2017-10-05 | 東ソー株式会社 | MODIFICATION TYPE RECOMBINANT FcγRIIb |
| WO2018056374A1 (en) * | 2016-09-23 | 2018-03-29 | 東ソー株式会社 | IMPROVED RECOMBINANT FcγRII |
| WO2018198817A1 (en) * | 2017-04-26 | 2018-11-01 | 東ソー株式会社 | Stable fc binding protein, method for producing said protein, and antibody adsorbent in which said protein is used |
| JP2019076069A (en) * | 2017-10-27 | 2019-05-23 | 東ソー株式会社 | Fc-binding proteins with amino acid substitution |
Also Published As
| Publication number | Publication date |
|---|---|
| JP7435939B2 (en) | 2024-02-21 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109476712B (en) | Novel alkali-stable immunoglobulin-binding proteins | |
| JP6883529B2 (en) | Methods and products for synthesizing fusion proteins | |
| US11926854B2 (en) | Soluble intein fusion proteins and methods for purifying biomolecules | |
| US9353161B2 (en) | Streptavidin mutein exhibiting reversible binding for biotin and streptavidin binding peptide tagged proteins | |
| JP2023119596A (en) | Fc binding proteins with cysteine in c terminal helical region | |
| JP2023507268A (en) | Binding protein of the enzyme acid alpha-glucosidase (GAA) and uses thereof | |
| WO2020099442A9 (en) | Novel triple-helical polypeptides lacking binding affinity for the fc domain of immunoglobulin and uses thereof | |
| JP2017178908A (en) | MODIFICATION TYPE RECOMBINANT FcγRIIb | |
| JP7435939B2 (en) | Highly productive Fc-binding protein and method for producing the same | |
| JP7386647B2 (en) | Fucose-binding protein with improved affinity for sugar chains and method for producing the same | |
| US9573982B2 (en) | Modified tamavidin | |
| JP6828291B2 (en) | A polynucleotide encoding human FcRn and a method for producing human FcRn using the polynucleotide. | |
| JP2022067620A (en) | Fucose-binding protein having improved heat stability, and method for producing the same | |
| JP2012130294A (en) | Antibody-binding protein and method for producing the same | |
| JP2014027916A (en) | Improved fc-binding protein and method for producing the same | |
| JP2015035958A (en) | POLYNUCLEOTIDE ENCODING HUMAN Fc RECEPTOR AND METHOD FOR PRODUCING HUMAN Fc RECEPTOR UTILIZING POLYNUCLEOTIDE CONCERNED | |
| JP2023141297A (en) | Highly thermostable fucose-binding protein and method for producing the same | |
| JP2021073877A (en) | Fc-binding proteins with improved acid stability, production methods thereof and antibody-adsorbing agents using same | |
| JP2020028285A (en) | Fc binding protein with improved acid stability, method for producing protein and antibody adsorbent using protein | |
| JP2017212902A (en) | POLYNUCLEOTIDE ENCODING FCγRIIA AND METHOD FOR PRODUCING FCγRIIA |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A821 Effective date: 20191111 |
|
| A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20221013 |
|
| A977 | Report on retrieval |
Free format text: JAPANESE INTERMEDIATE CODE: A971007 Effective date: 20230823 |
|
| A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20230919 |
|
| A601 | Written request for extension of time |
Free format text: JAPANESE INTERMEDIATE CODE: A601 Effective date: 20231113 |
|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20231120 |
|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20231205 |
|
| TRDD | Decision of grant or rejection written | ||
| A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 Effective date: 20240109 |
|
| A61 | First payment of annual fees (during grant procedure) |
Free format text: JAPANESE INTERMEDIATE CODE: A61 Effective date: 20240123 |
|
| R150 | Certificate of patent or registration of utility model |
Ref document number: 7435939 Country of ref document: JP Free format text: JAPANESE INTERMEDIATE CODE: R150 |