JP6303195B2 - Method for producing functional foreign protein by bacteria - Google Patents
Method for producing functional foreign protein by bacteria Download PDFInfo
- Publication number
- JP6303195B2 JP6303195B2 JP2013240294A JP2013240294A JP6303195B2 JP 6303195 B2 JP6303195 B2 JP 6303195B2 JP 2013240294 A JP2013240294 A JP 2013240294A JP 2013240294 A JP2013240294 A JP 2013240294A JP 6303195 B2 JP6303195 B2 JP 6303195B2
- Authority
- JP
- Japan
- Prior art keywords
- protein
- scfv
- zif268
- expression
- dna binding
- 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.)
- Active
Links
- 108090000623 proteins and genes Proteins 0.000 title claims description 168
- 102000004169 proteins and genes Human genes 0.000 title claims description 134
- 241000894006 Bacteria Species 0.000 title claims description 17
- 238000004519 manufacturing process Methods 0.000 title description 10
- 230000014509 gene expression Effects 0.000 claims description 99
- 210000000805 cytoplasm Anatomy 0.000 claims description 76
- 238000000034 method Methods 0.000 claims description 68
- 230000004568 DNA-binding Effects 0.000 claims description 53
- 239000013604 expression vector Substances 0.000 claims description 40
- 108020001507 fusion proteins Proteins 0.000 claims description 37
- 102000037865 fusion proteins Human genes 0.000 claims description 35
- 101150006320 trpR gene Proteins 0.000 claims description 33
- 150000001413 amino acids Chemical class 0.000 claims description 28
- 230000001580 bacterial effect Effects 0.000 claims description 24
- 210000004899 c-terminal region Anatomy 0.000 claims description 24
- 102000052510 DNA-Binding Proteins Human genes 0.000 claims description 23
- 101710096438 DNA-binding protein Proteins 0.000 claims description 22
- 108020004707 nucleic acids Proteins 0.000 claims description 21
- 102000039446 nucleic acids Human genes 0.000 claims description 21
- 150000007523 nucleic acids Chemical class 0.000 claims description 21
- 230000002708 enhancing effect Effects 0.000 claims description 17
- 230000001086 cytosolic effect Effects 0.000 claims description 13
- 102000008394 Immunoglobulin Fragments Human genes 0.000 claims description 10
- 108010021625 Immunoglobulin Fragments Proteins 0.000 claims description 10
- 102000004190 Enzymes Human genes 0.000 claims description 6
- 108090000790 Enzymes Proteins 0.000 claims description 6
- -1 Zif268 Proteins 0.000 claims description 3
- 239000003623 enhancer Substances 0.000 claims description 3
- 230000008569 process Effects 0.000 claims description 3
- 239000004480 active ingredient Substances 0.000 claims description 2
- 125000003275 alpha amino acid group Chemical group 0.000 claims 12
- 235000018102 proteins Nutrition 0.000 description 98
- 241000588724 Escherichia coli Species 0.000 description 87
- 230000027455 binding Effects 0.000 description 36
- 210000001322 periplasm Anatomy 0.000 description 36
- 239000012634 fragment Substances 0.000 description 34
- 239000013598 vector Substances 0.000 description 27
- 230000000694 effects Effects 0.000 description 25
- 239000000427 antigen Substances 0.000 description 17
- 102000036639 antigens Human genes 0.000 description 17
- 108091007433 antigens Proteins 0.000 description 17
- 238000002965 ELISA Methods 0.000 description 16
- 101710175625 Maltose/maltodextrin-binding periplasmic protein Proteins 0.000 description 15
- 241000287828 Gallus gallus Species 0.000 description 14
- 210000004027 cell Anatomy 0.000 description 14
- 239000013612 plasmid Substances 0.000 description 14
- 238000002360 preparation method Methods 0.000 description 13
- 108020004414 DNA Proteins 0.000 description 12
- 108060001084 Luciferase Proteins 0.000 description 12
- 239000005089 Luciferase Substances 0.000 description 11
- 230000001976 improved effect Effects 0.000 description 10
- 241000963438 Gaussia <copepod> Species 0.000 description 9
- 241000699666 Mus <mouse, genus> Species 0.000 description 9
- 238000012300 Sequence Analysis Methods 0.000 description 9
- 238000000246 agarose gel electrophoresis Methods 0.000 description 9
- 108010076504 Protein Sorting Signals Proteins 0.000 description 8
- 235000001014 amino acid Nutrition 0.000 description 8
- 239000006166 lysate Substances 0.000 description 7
- 230000009257 reactivity Effects 0.000 description 7
- 108010006519 Molecular Chaperones Proteins 0.000 description 6
- 238000012181 QIAquick gel extraction kit Methods 0.000 description 6
- 238000006243 chemical reaction Methods 0.000 description 6
- 230000004927 fusion Effects 0.000 description 6
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 6
- 238000005259 measurement Methods 0.000 description 6
- 239000002609 medium Substances 0.000 description 6
- 238000000746 purification Methods 0.000 description 6
- 230000003248 secreting effect Effects 0.000 description 6
- 101100136076 Aspergillus oryzae (strain ATCC 42149 / RIB 40) pel1 gene Proteins 0.000 description 5
- 108010056307 Hin recombinase Proteins 0.000 description 5
- 108010052285 Membrane Proteins Proteins 0.000 description 5
- 102000018697 Membrane Proteins Human genes 0.000 description 5
- 241000283973 Oryctolagus cuniculus Species 0.000 description 5
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 5
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 5
- 238000005119 centrifugation Methods 0.000 description 5
- 230000006870 function Effects 0.000 description 5
- 239000000499 gel Substances 0.000 description 5
- 230000001939 inductive effect Effects 0.000 description 5
- 101150040383 pel2 gene Proteins 0.000 description 5
- 101150050446 pelB gene Proteins 0.000 description 5
- 238000011084 recovery Methods 0.000 description 5
- 239000006228 supernatant Substances 0.000 description 5
- 239000011701 zinc Substances 0.000 description 5
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 4
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 4
- 241000124008 Mammalia Species 0.000 description 4
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 4
- 229960000723 ampicillin Drugs 0.000 description 4
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- 229940088598 enzyme Drugs 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 210000003000 inclusion body Anatomy 0.000 description 4
- 244000005700 microbiome Species 0.000 description 4
- 238000002823 phage display Methods 0.000 description 4
- 230000009466 transformation Effects 0.000 description 4
- 230000007704 transition Effects 0.000 description 4
- 241000238421 Arthropoda Species 0.000 description 3
- 241000194107 Bacillus megaterium Species 0.000 description 3
- 108700034853 E coli TRPR Proteins 0.000 description 3
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 3
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 3
- 102100025169 Max-binding protein MNT Human genes 0.000 description 3
- 102000005431 Molecular Chaperones Human genes 0.000 description 3
- 108091028043 Nucleic acid sequence Proteins 0.000 description 3
- 108091005804 Peptidases Proteins 0.000 description 3
- 229920001213 Polysorbate 20 Polymers 0.000 description 3
- 239000004365 Protease Substances 0.000 description 3
- 229940124158 Protease/peptidase inhibitor Drugs 0.000 description 3
- 102000013090 Thioredoxin-Disulfide Reductase Human genes 0.000 description 3
- 108010079911 Thioredoxin-disulfide reductase Proteins 0.000 description 3
- 238000002835 absorbance Methods 0.000 description 3
- MGSKVZWGBWPBTF-UHFFFAOYSA-N aebsf Chemical compound NCCC1=CC=C(S(F)(=O)=O)C=C1 MGSKVZWGBWPBTF-UHFFFAOYSA-N 0.000 description 3
- 230000008827 biological function Effects 0.000 description 3
- 238000006664 bond formation reaction Methods 0.000 description 3
- 238000012258 culturing Methods 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 238000000605 extraction Methods 0.000 description 3
- 230000001965 increasing effect Effects 0.000 description 3
- 239000012528 membrane Substances 0.000 description 3
- 230000035772 mutation Effects 0.000 description 3
- 230000001590 oxidative effect Effects 0.000 description 3
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 3
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 3
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 230000028327 secretion Effects 0.000 description 3
- 108091006107 transcriptional repressors Proteins 0.000 description 3
- 238000011426 transformation method Methods 0.000 description 3
- DLZKEQQWXODGGZ-KCJUWKMLSA-N 2-[[(2r)-2-[[(2s)-2-amino-3-(4-hydroxyphenyl)propanoyl]amino]propanoyl]amino]acetic acid Chemical compound OC(=O)CNC(=O)[C@@H](C)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 DLZKEQQWXODGGZ-KCJUWKMLSA-N 0.000 description 2
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 2
- HRPVXLWXLXDGHG-UHFFFAOYSA-N Acrylamide Chemical compound NC(=O)C=C HRPVXLWXLXDGHG-UHFFFAOYSA-N 0.000 description 2
- 235000014469 Bacillus subtilis Nutrition 0.000 description 2
- 108700020911 DNA-Binding Proteins Proteins 0.000 description 2
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 2
- 102100039556 Galectin-4 Human genes 0.000 description 2
- 239000004471 Glycine Substances 0.000 description 2
- 101000608765 Homo sapiens Galectin-4 Proteins 0.000 description 2
- 101000825161 Homo sapiens Transcription factor Spi-C Proteins 0.000 description 2
- 101150028955 MBP gene Proteins 0.000 description 2
- 241000282553 Macaca Species 0.000 description 2
- 108010014251 Muramidase Proteins 0.000 description 2
- 102000016943 Muramidase Human genes 0.000 description 2
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 description 2
- PXHVJJICTQNCMI-UHFFFAOYSA-N Nickel Chemical compound [Ni] PXHVJJICTQNCMI-UHFFFAOYSA-N 0.000 description 2
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 2
- 241000607142 Salmonella Species 0.000 description 2
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 2
- 229930006000 Sucrose Natural products 0.000 description 2
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 2
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 description 2
- 238000001042 affinity chromatography Methods 0.000 description 2
- 125000000539 amino acid group Chemical group 0.000 description 2
- 210000004102 animal cell Anatomy 0.000 description 2
- 108010051210 beta-Fructofuranosidase Proteins 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 101150017872 clpQ gene Proteins 0.000 description 2
- 239000011248 coating agent Substances 0.000 description 2
- 238000000576 coating method Methods 0.000 description 2
- 239000000287 crude extract Substances 0.000 description 2
- 238000012217 deletion Methods 0.000 description 2
- 230000037430 deletion Effects 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 238000005194 fractionation Methods 0.000 description 2
- 101150055178 hslV gene Proteins 0.000 description 2
- 230000002209 hydrophobic effect Effects 0.000 description 2
- 230000016784 immunoglobulin production Effects 0.000 description 2
- 230000003834 intracellular effect Effects 0.000 description 2
- 239000001573 invertase Substances 0.000 description 2
- 235000011073 invertase Nutrition 0.000 description 2
- 238000004255 ion exchange chromatography Methods 0.000 description 2
- 229960000274 lysozyme Drugs 0.000 description 2
- 239000004325 lysozyme Substances 0.000 description 2
- 235000010335 lysozyme Nutrition 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 230000000813 microbial effect Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 108090000765 processed proteins & peptides Proteins 0.000 description 2
- 238000001742 protein purification Methods 0.000 description 2
- 102000005962 receptors Human genes 0.000 description 2
- 108020003175 receptors Proteins 0.000 description 2
- 238000003259 recombinant expression Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 101150034869 rpo5 gene Proteins 0.000 description 2
- 101150106872 rpoH gene Proteins 0.000 description 2
- 238000005185 salting out Methods 0.000 description 2
- 235000020183 skimmed milk Nutrition 0.000 description 2
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 239000005720 sucrose Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 238000013518 transcription Methods 0.000 description 2
- 230000035897 transcription Effects 0.000 description 2
- 230000001131 transforming effect Effects 0.000 description 2
- 238000001262 western blot Methods 0.000 description 2
- 229910052725 zinc Inorganic materials 0.000 description 2
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 description 1
- 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 description 1
- 241000271566 Aves Species 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 244000063299 Bacillus subtilis Species 0.000 description 1
- 241000149420 Bothrometopus brevis Species 0.000 description 1
- 125000001433 C-terminal amino-acid group Chemical group 0.000 description 1
- 241000218645 Cedrus Species 0.000 description 1
- 108020004705 Codon Proteins 0.000 description 1
- BWGNESOTFCXPMA-UHFFFAOYSA-N Dihydrogen disulfide Chemical compound SS BWGNESOTFCXPMA-UHFFFAOYSA-N 0.000 description 1
- 101150114117 EGR1 gene Proteins 0.000 description 1
- 108010013369 Enteropeptidase Proteins 0.000 description 1
- 102100029727 Enteropeptidase Human genes 0.000 description 1
- 102000003951 Erythropoietin Human genes 0.000 description 1
- 108090000394 Erythropoietin Proteins 0.000 description 1
- 241001198387 Escherichia coli BL21(DE3) Species 0.000 description 1
- 241000206602 Eukaryota Species 0.000 description 1
- 108010074860 Factor Xa Proteins 0.000 description 1
- 108010063907 Glutathione Reductase Proteins 0.000 description 1
- 102100036442 Glutathione reductase, mitochondrial Human genes 0.000 description 1
- 102000004269 Granulocyte Colony-Stimulating Factor Human genes 0.000 description 1
- 108010017080 Granulocyte Colony-Stimulating Factor Proteins 0.000 description 1
- 108010051696 Growth Hormone Proteins 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 101001049697 Homo sapiens Early growth response protein 1 Proteins 0.000 description 1
- 108060003951 Immunoglobulin Proteins 0.000 description 1
- 102000017727 Immunoglobulin Variable Region Human genes 0.000 description 1
- 108010067060 Immunoglobulin Variable Region Proteins 0.000 description 1
- 108010050904 Interferons Proteins 0.000 description 1
- 102000014150 Interferons Human genes 0.000 description 1
- 102000004195 Isomerases Human genes 0.000 description 1
- 108090000769 Isomerases Proteins 0.000 description 1
- 241000186660 Lactobacillus Species 0.000 description 1
- 241000194036 Lactococcus Species 0.000 description 1
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 description 1
- 241000699660 Mus musculus Species 0.000 description 1
- 101100178822 Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv) htrA1 gene Proteins 0.000 description 1
- 241000588653 Neisseria Species 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 108010079246 OMPA outer membrane proteins Proteins 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 108010076181 Proinsulin Proteins 0.000 description 1
- 108020004511 Recombinant DNA Proteins 0.000 description 1
- 101100277437 Rhizobium meliloti (strain 1021) degP1 gene Proteins 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 102000007562 Serum Albumin Human genes 0.000 description 1
- 108010071390 Serum Albumin Proteins 0.000 description 1
- 102100038803 Somatotropin Human genes 0.000 description 1
- 241000191940 Staphylococcus Species 0.000 description 1
- 244000057717 Streptococcus lactis Species 0.000 description 1
- 235000014897 Streptococcus lactis Nutrition 0.000 description 1
- 239000004098 Tetracycline Substances 0.000 description 1
- 102000002933 Thioredoxin Human genes 0.000 description 1
- 108090000190 Thrombin Proteins 0.000 description 1
- 102000036693 Thrombopoietin Human genes 0.000 description 1
- 108010041111 Thrombopoietin Proteins 0.000 description 1
- AUYYCJSJGJYCDS-LBPRGKRZSA-N Thyrolar Chemical class IC1=CC(C[C@H](N)C(O)=O)=CC(I)=C1OC1=CC=C(O)C(I)=C1 AUYYCJSJGJYCDS-LBPRGKRZSA-N 0.000 description 1
- 108090000373 Tissue Plasminogen Activator Proteins 0.000 description 1
- 102000003978 Tissue Plasminogen Activator Human genes 0.000 description 1
- 108091023040 Transcription factor Proteins 0.000 description 1
- 102000040945 Transcription factor Human genes 0.000 description 1
- 102100022285 Transcription factor Spi-C Human genes 0.000 description 1
- 101710154918 Trigger factor Proteins 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- PTFCDOFLOPIGGS-UHFFFAOYSA-N Zinc dication Chemical compound [Zn+2] PTFCDOFLOPIGGS-UHFFFAOYSA-N 0.000 description 1
- 101710185494 Zinc finger protein Proteins 0.000 description 1
- 102100023597 Zinc finger protein 816 Human genes 0.000 description 1
- 238000007792 addition Methods 0.000 description 1
- PYMYPHUHKUWMLA-VAYJURFESA-N aldehydo-L-arabinose Chemical compound OC[C@H](O)[C@H](O)[C@@H](O)C=O PYMYPHUHKUWMLA-VAYJURFESA-N 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 description 1
- 239000013602 bacteriophage vector Substances 0.000 description 1
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 210000005056 cell body Anatomy 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 230000010307 cell transformation Effects 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 239000003593 chromogenic compound Substances 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 230000004186 co-expression Effects 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 239000013601 cosmid vector Substances 0.000 description 1
- 101150110403 cspA gene Proteins 0.000 description 1
- 125000000151 cysteine group Chemical group N[C@@H](CS)C(=O)* 0.000 description 1
- 230000003013 cytotoxicity Effects 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
- 101150018266 degP gene Proteins 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 238000006471 dimerization reaction Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 230000001516 effect on protein Effects 0.000 description 1
- 230000005611 electricity Effects 0.000 description 1
- 238000004520 electroporation Methods 0.000 description 1
- 230000009881 electrostatic interaction Effects 0.000 description 1
- 229940105423 erythropoietin Drugs 0.000 description 1
- 210000003527 eukaryotic cell Anatomy 0.000 description 1
- 230000005714 functional activity Effects 0.000 description 1
- 238000001641 gel filtration chromatography Methods 0.000 description 1
- 238000002523 gelfiltration Methods 0.000 description 1
- 238000007429 general method Methods 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 239000000122 growth hormone Substances 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 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 1
- 102000018358 immunoglobulin Human genes 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 239000000411 inducer Substances 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 229940079322 interferon Drugs 0.000 description 1
- 102000002467 interleukin receptors Human genes 0.000 description 1
- 108010093036 interleukin receptors Proteins 0.000 description 1
- 229940039696 lactobacillus Drugs 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 238000004020 luminiscence type Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 229910052759 nickel Inorganic materials 0.000 description 1
- 102000006255 nuclear receptors Human genes 0.000 description 1
- 108020004017 nuclear receptors Proteins 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 101150093139 ompT gene Proteins 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 230000000704 physical effect Effects 0.000 description 1
- 239000013600 plasmid vector Substances 0.000 description 1
- 101150052371 plsX gene Proteins 0.000 description 1
- OXCMYAYHXIHQOA-UHFFFAOYSA-N potassium;[2-butyl-5-chloro-3-[[4-[2-(1,2,4-triaza-3-azanidacyclopenta-1,4-dien-5-yl)phenyl]phenyl]methyl]imidazol-4-yl]methanol Chemical compound [K+].CCCCC1=NC(Cl)=C(CO)N1CC1=CC=C(C=2C(=CC=CC=2)C2=N[N-]N=N2)C=C1 OXCMYAYHXIHQOA-UHFFFAOYSA-N 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000002731 protein assay Methods 0.000 description 1
- 230000012846 protein folding Effects 0.000 description 1
- 238000000164 protein isolation Methods 0.000 description 1
- 210000001938 protoplast Anatomy 0.000 description 1
- 230000008844 regulatory mechanism Effects 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 238000004007 reversed phase HPLC Methods 0.000 description 1
- 239000012723 sample buffer Substances 0.000 description 1
- 108010073863 saruplase Proteins 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 238000005549 size reduction Methods 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 239000003270 steroid hormone Substances 0.000 description 1
- 150000003431 steroids Chemical class 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 229960002180 tetracycline Drugs 0.000 description 1
- 229930101283 tetracycline Natural products 0.000 description 1
- 235000019364 tetracycline Nutrition 0.000 description 1
- 150000003522 tetracyclines Chemical class 0.000 description 1
- 238000010257 thawing Methods 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 238000009210 therapy by ultrasound Methods 0.000 description 1
- 108060008226 thioredoxin Proteins 0.000 description 1
- 229940094937 thioredoxin Drugs 0.000 description 1
- 229960004072 thrombin Drugs 0.000 description 1
- 239000005495 thyroid hormone Substances 0.000 description 1
- 229940036555 thyroid hormone Drugs 0.000 description 1
- 229960000187 tissue plasminogen activator Drugs 0.000 description 1
- 230000005026 transcription initiation Effects 0.000 description 1
- 230000002103 transcriptional effect Effects 0.000 description 1
- 230000002463 transducing effect Effects 0.000 description 1
- 239000012581 transferrin Substances 0.000 description 1
- 230000005945 translocation Effects 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
- 238000002525 ultrasonication Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Description
本発明は、細菌の細胞質内で、所望の外来タンパク質、特に組換え単鎖抗体（scFv）を、生物学的な機能を保持し、可溶性の状態で効率よく発現させることで、大量生産する方法に関する。 The present invention provides a method for mass production of a desired foreign protein, particularly a recombinant single chain antibody (scFv), in a bacterial cytoplasm by efficiently expressing it in a soluble state while retaining its biological function. About.
抗体は、研究、検査、医薬などの分野で無くてはならない存在であるが、近年、抗体医薬としてより重要性が高まっている。これには、組換え技術によるマウス抗体のヒト化技術が大きく貢献し、ファージディスプレイ技術と合わせて、新しい抗体作製技術が大きく進展している。 Antibodies are indispensable in the fields of research, testing, medicine and the like, but in recent years, they have become more important as antibody medicines. To this end, the humanization technology of mouse antibodies by recombinant technology has greatly contributed, and new antibody production technologies have been greatly developed together with the phage display technology.
組換え抗体は、従来の抗体に、親和性の増強、安定性の増強、他のタンパク質との融合、細胞内での発現、小サイズ化、多量体化等の性質を持たせることができる。形態は、IgGと同様なもの、IgGからFc部分を取り除いたFab、抗体認識部分のみのscFv（単鎖抗体）等がある。その中でもscFvは、分子量が25kDa程度とIgGの分子量の6分の1程と小さい。抗体としての結合能は、IgGと全く同様でありながら、小サイズのため取扱いが大変容易であり、研究・検査分野において多くの利用が期待されている。細胞への浸潤性がよいことから、がん治療のツールとしても有望視されている。 Recombinant antibodies can impart properties such as enhanced affinity, enhanced stability, fusion with other proteins, intracellular expression, size reduction, and multimerization to conventional antibodies. The form includes the same as IgG, Fab obtained by removing the Fc portion from IgG, scFv (single chain antibody) having only the antibody recognition portion, and the like. Among them, scFv has a molecular weight of about 25 kDa, which is as small as 1/6 of the molecular weight of IgG. The binding ability as an antibody is exactly the same as that of IgG, but it is very easy to handle because of its small size, and many uses are expected in the field of research and testing. It is promising as a tool for cancer treatment because of its good invasiveness to cells.
組換え抗体は、現在おもに培養動物細胞を用いて発現されている。培養動物細胞を用いることで、機能的な抗体（抗原認識機能を持つ）を発現させることができるが、その発現には、長期間を要し、コストも大きく、組換え抗体製品が高価なことの原因となっている。微生物を用いれば、大量にスピーディーに発現が可能となる。大腸菌は増殖が速く培養が容易なため、組換えタンパク質の大量発現系の宿主として汎用されている。しかし、組換えタンパク質が大腸菌体内で封入体を形成してしまい、他の発現系が必要となる場合も多い。scFvもそのタンパク質としての性質から、大腸菌で発現させようとすると、多くの場合封入体を形成し、可溶化した状態で発現できないのが現状である。 Recombinant antibodies are currently expressed primarily using cultured animal cells. By using cultured animal cells, functional antibodies (with antigen recognition function) can be expressed, but the expression takes a long time, is expensive, and the recombinant antibody product is expensive. Cause. If microorganisms are used, it becomes possible to express rapidly in large quantities. Escherichia coli is widely used as a host for recombinant protein mass expression systems because it grows quickly and is easy to culture. However, the recombinant protein often forms inclusion bodies in E. coli, and other expression systems are often required. Due to the nature of the protein, scFv is often unable to be expressed in a solubilized state in many cases when it is expressed in E. coli.
大腸菌において、scFvの発現を試みると、scFvの種類を問わず、ほとんどの場合は細胞内で封入体として沈殿してしまい、機能的なタンパク質として回収できない（非特許文献１）。これは、scFvが真核細胞由来のタンパク質であるという本質的な理由による。抗体をはじめ、真核生物由来のタンパク質の多くはジスルフィド結合を有している。scFvにも複数のジスルフィド結合が存在している。大腸菌の細胞質内は強い還元条件下にあるため、真核生物由来のタンパク質を発現させても正しいジスルフィド結合を形成し本来の立体構造を再現するのはscFvのみならず、一般的にも困難である。大腸菌内で発現した外来タンパク質が本来の立体構造とは異なると、封入体を形成して不溶化してしまう場合だけでなく、細胞質内のプロテアーゼで分解されてしまったり、細胞毒性を示し宿主大腸菌の生育が悪くなるなどの問題が発生する。 In E. coli, when scFv expression is attempted, regardless of the type of scFv, in most cases, it precipitates as inclusion bodies in the cell and cannot be recovered as a functional protein (Non-patent Document 1). This is for the essential reason that scFv is a protein derived from a eukaryotic cell. Many eukaryotic proteins, including antibodies, have disulfide bonds. There are multiple disulfide bonds in scFv. Since the cytoplasm of E. coli is under strong reducing conditions, it is difficult not only to scFv, but also to reproduce the original three-dimensional structure even when expressing eukaryotic proteins. is there. If the foreign protein expressed in E. coli is different from the original three-dimensional structure, it will not only form inclusion bodies and become insolubilized, but it may be degraded by proteases in the cytoplasm, exhibit cytotoxicity and Problems such as poor growth occur.
このような外来タンパク質を大腸菌宿主で効率よく発現させるための方法としては、ジスルフィド結合の形成に適した酸化的環境にあるペリプラズムで発現する方法が提案されている（非特許文献３）。大腸菌のジスルフィド結合形成に関与し、シャペロン機能も有するタンパク質であるDsbAなどの各種Dsbファミリータンパク質は、ペリプラズム空間に偏在しているため、ペリプラズム移行シグナルを外来タンパク質に付加し、ペリプラズム領域内に分泌・発現させることで、機能的な組換え外来タンパク質を回収することができる場合が多い（非特許文献４）。さらに、DsbAなどのDsbファミリータンパク質を発現する発現ベクターを、外来タンパク質をペリプラズム領域内で分泌・発現可能な発現ベクターと共発現させて、正しいジスルフィド結合能を強化する方法も提案されている（特許文献１）。また、リフォールディング促進活性が知られているトリガーファクターを発現する発現ベクターを、外来タンパク質の発現ベクターと共発現させる方法も提案されている（特許文献２）。これらの手法はscFvに適用することも可能で、ペリプラズム領域内であれば機能的なscFvを回収することができる。ペリプラズム領域内で機能的なscFvを得る際に、scFvをタンデムscFv（scFv-scFv）結合体、又は２量体化能のあるロイシンジッパードメインなどの多量体化能を有するタンパク質との融合タンパクとして発現させてscFv多量体とすることで、機能的scFvの抗原との反応性を増大させる方法（非特許文献１１、１４）、さらに当該手法をファージディスプレイ法に応用して、機能的なscFv多量体をファージ上にディスプレイし、ハイスループットな抗原特異的scFvを選択する方法も開発されている（非特許文献１２）。しかしながら、狭いペリプラズム空間で発現させる方法では、機能的なscFvは得られるものの、発現タンパク質としての回収率が低く、本来の目的である細菌を用いて高発現させるという目的は達せられない。 As a method for efficiently expressing such a foreign protein in an E. coli host, a method for expressing it in the periplasm in an oxidative environment suitable for disulfide bond formation has been proposed (Non-patent Document 3). Since various Dsb family proteins such as DsbA, which are involved in the formation of disulfide bonds in E. coli and also have chaperone functions, are ubiquitous in the periplasmic space, they add periplasmic transition signals to foreign proteins and are secreted into the periplasmic region. In many cases, functional recombinant foreign proteins can be recovered by expression (Non-patent Document 4). Furthermore, a method has also been proposed in which an expression vector that expresses a Dsb family protein such as DsbA is co-expressed with an expression vector capable of secreting and expressing a foreign protein in the periplasmic region to enhance the correct disulfide bondability (patent) Reference 1). In addition, a method has been proposed in which an expression vector that expresses a trigger factor known to have refolding promoting activity is co-expressed with an expression vector of a foreign protein (Patent Document 2). These methods can also be applied to scFv, and functional scFv can be recovered within the periplasm region. When obtaining a functional scFv in the periplasmic region, the scFv is fused with a protein having multimerization ability such as a tandem scFv (scFv-scFv) conjugate or a leucine zipper domain capable of dimerization. A method of increasing the reactivity of functional scFv with an antigen by expressing it as an scFv multimer (Non-patent Documents 11 and 14), and further applying this technique to the phage display method, A method of displaying a body on a phage and selecting a high-throughput antigen-specific scFv has also been developed (Non-patent Document 12). However, in the method of expressing in a narrow periplasmic space, a functional scFv can be obtained, but the recovery rate as an expressed protein is low, and the purpose of high expression using bacteria, which is the original purpose, cannot be achieved.
一方、細胞質においてジスルフィド結合を形成し正しい立体構造のタンパク質を発現するための手法として、細胞質内環境の改良が施された大腸菌ホスト株（コンピテントセル）が開発されている。市販されている主な菌株としては、New England Biolabs社からの「SHuffleコンピテントセル」及びNovagen社からの「Origamiコンピテントセル」がある。前者においてはジスルフィド結合形成を妨げるグルタチオンリダクターゼ（Gor）やチオレドキシンリダクターゼ（TreR）が不活性化され、さらに、本来ペリプラズムに存在して、正しいジスルフィド結合の形成能及びシャペロン機能を有するジスルフィド結合イソメラーゼ（DsbC）が細胞質で発現するように改良されている。後者においては、TreRとGorに変異をもたせることで、細胞質でのジスルフィド結合形成能を向上させ、細胞質内でのタンパク質フォールディングが可能となるように改良されている。このような細胞質環境が改変された特殊な大腸菌を用いた場合には、scFvについても、細胞質内で機能的scFvの発現に成功している（非特許文献５、１５など）が、発現量は野生型大腸菌でのペリプラズム内での発現量を超えるまでには至っていないようである（非特許文献６）。 On the other hand, E. coli host strains (competent cells) with an improved cytoplasmic environment have been developed as a method for expressing disulfide bonds in the cytoplasm and expressing proteins with the correct three-dimensional structure. The main strains on the market are “SHuffle competent cells” from New England Biolabs and “Origami competent cells” from Novagen. In the former, glutathione reductase (Gor) and thioredoxin reductase (TreR), which prevent disulfide bond formation, are inactivated, and disulfide bond isomerase (DsbC) that originally exists in the periplasm and has the correct disulfide bond-forming ability and chaperone function. ) Has been improved to be expressed in the cytoplasm. In the latter, the mutation is made in TreR and Gor, so that the ability to form disulfide bonds in the cytoplasm is improved, and protein folding in the cytoplasm is enabled. When such special E. coli having a modified cytoplasmic environment is used, scFv has also been successfully expressed in the cytoplasm as a functional scFv (Non-Patent Documents 5, 15 etc.), but the expression level is It does not seem to reach the expression level in the periplasm in wild-type E. coli (Non-patent Document 6).
一般に、タンパク質発現が大腸菌等でうまくいかない時は、親水性の高いタンパク質と融合させて発現させることで成功する例が多く（非特許文献７）、ビオチン化ドメインを備える親水性タグと融合させて発現を改善する例（特許文献３）も知られている。scFv発現に関しても多くの試みがなされている。scFvに対し、親水性の高いマルトース結合タンパク質を融合させて、分子全体の親水性を上げて発現量を上げた旨の報告もある（非特許文献８）。なお、この報告では、マルトース結合タンパク質-scFvの細胞質発現量が（200mg/L, 培養液の600nm吸光度が2の時）とされ、あまりの発現量の大きさに注目を集めたが、追試実験での再現性も含め、このシステムを用いた続報はない。しかも、得られたscFvに対して定量的な抗原結合活性実験が行われていない。
また、scFvを酵母GAL4由来のDNA結合ドメインとの融合タンパク質を発現するベクターを構築し、一般的な大腸菌宿主の細胞質内で該融合タンパク質を発現させたことの報告があるが、90％程度がインクルージョンボディに含まれている状態での発現であり、細胞質内での機能的scFv発現に成功したとまではいえない（非特許文献９）。
最近、scFvをマルトース結合タンパク質やチオレドキシンのような親水性の高いタンパク質との融合体とした上で、細胞質内を酸化的環境に改良した改変大腸菌を用いてその細胞質内で発現させ、かつ界面活性剤を加えた溶媒中で回収することにより、溶解性が高い機能的scFvを得たという報告がある（非特許文献１６）が、多段階の煩雑な工程が必要である。
In general, when protein expression is not successful in Escherichia coli, etc., there are many examples of success by expressing it by fusing with a highly hydrophilic protein (Non-patent Document 7), and fusing with a hydrophilic tag having a biotinylated domain. An example of improving the above (Patent Document 3) is also known. Many attempts have also been made regarding scFv expression. There is also a report that the expression level is increased by fusing scFv with a highly hydrophilic maltose binding protein to increase the hydrophilicity of the whole molecule (Non-patent Document 8). In this report, the cytoplasmic expression level of maltose-binding protein-scFv was set to 200 mg / L (when the absorbance at 600 nm of the culture solution was 2). There is no follow-up using this system, including reproducibility in Moreover, quantitative antigen binding activity experiments have not been performed on the obtained scFv.
In addition, it has been reported that a vector expressing a fusion protein of scFv and a DNA binding domain derived from yeast GAL4 was constructed, and the fusion protein was expressed in the cytoplasm of a general E. coli host. It is an expression in the state of being included in the inclusion body, and it cannot be said that the functional scFv expression in the cytoplasm was successful (Non-patent Document 9).
Recently, scFv was made into a fusion with a highly hydrophilic protein such as maltose-binding protein or thioredoxin, expressed in the cytoplasm using modified Escherichia coli with an improved oxidative environment in the cytoplasm, and surface activity. There is a report that functional scFv having high solubility was obtained by collecting in a solvent to which an agent was added (Non-patent Document 16), but a complicated multi-step process is required.
このように、一般的な大腸菌宿主を用いて細胞質内で機能的scFvを大量に発現させようとする試みは未だ成功しているとは言い難く、発現量が増える場合には機能的ではないタンパク質、すなわち変性した抗原結合能の弱いscFvしか回収されず（非特許文献６）、scFvの細胞質内での発現の問題点の大きな原因は、細胞質内が還元的環境下であることとされる（非特許文献１３）。その結果、scFvの発現については、収量は低くなってしまうものの、ペリプラズムでの発現が重要であることが広く認められている（非特許文献１、１３）。 Thus, attempts to express a large amount of functional scFv in the cytoplasm using a general E. coli host are not yet successful, and proteins that are not functional when the expression level increases That is, only a modified scFv having a weak antigen-binding ability is recovered (Non-patent Document 6), and the major cause of the problem of expression of scFv in the cytoplasm is that the cytoplasm is in a reducing environment ( Non-patent document 13). As a result, regarding the expression of scFv, although the yield is low, it is widely accepted that expression in the periplasm is important (Non-patent Documents 1 and 13).
また、発現したタンパク質を菌体外に分泌することのできるBacillus megateriumを用いて、機能的なscFvを培養液内に分泌生産する方法も提唱されているが（非特許文献１０）、多くの成功例が報告されるには至っていない。分泌システムを利用した発現は、scFv内のジスルフィド結合は正常に結ばれることが期待されるが、大量の培養液中にscFvが存在するので、濃縮の作業が必要になる点は大きなデメリットである。これに対して、細菌の細胞質内で正常なscFvを発現できれば、宿主菌を遠心後、非常に濃縮された状態で精製する事ができ、大量の培養液濃縮工程の手間がかからず簡便である。
以上のことから、有用性の高いscFvを正常な可溶化状態で、一般的な細菌の細胞質内において安価に大量生産させる手法が、待ち望まれていた。
In addition, a method of secreting and producing functional scFv into the culture solution using Bacillus megaterium capable of secreting the expressed protein outside the cell has been proposed (Non-patent Document 10), but many successes have been proposed. No examples have been reported. Expression using the secretory system is expected to cause normal disulfide bonds in scFv, but scFv is present in a large volume of culture medium, so the concentration work is a major disadvantage. . On the other hand, if normal scFv can be expressed in the cytoplasm of the bacteria, the host bacteria can be purified in a highly concentrated state after centrifugation. is there.
In view of the above, a method for mass-producing a highly useful scFv in a normal solubilized state in a general bacterial cytoplasm at low cost has been awaited.
背景技術に示したように、一般的な細菌宿主、特に大腸菌宿主を用いて真核生物由来の所望の外来タンパク質の生物学的機能を保持した状態の機能的タンパク質を細胞質内に高発現させる、又は正常な立体構造（本来の折りたたみ状態）を保持した可溶性状態で細胞質内に高発現させる方法であって、scFvに対しても適用可能な方法の提供が強く望まれていた。なお、本発明において、「機能的タンパク質」というとき、「本来の生物学的機能を保持した組換えタンパク質」を指すが、そのような組換えタンパク質は、通常、タンパク質本来の正常な立体構造（折りたたみ状態）を保持し、かつ可溶性状態にある組換えタンパク質でもあるので、後者の意味で用いることもある。 As shown in the background art, using a general bacterial host, in particular, an E. coli host, a functional protein in a state that retains the biological function of a desired eukaryotic-derived foreign protein is highly expressed in the cytoplasm. Alternatively, it has been strongly desired to provide a method that is highly expressed in the cytoplasm in a soluble state that retains a normal three-dimensional structure (original folded state) and can be applied to scFv. In the present invention, the term “functional protein” refers to a “recombinant protein that retains its original biological function”, and such a recombinant protein usually has a normal three-dimensional structure ( Since it is also a recombinant protein that retains a folded state and is in a soluble state, it may be used in the latter sense.
本発明者らは、この問題解決に鋭意取り組み、宿主大腸菌に対して特殊な改良を施さなくても、一般的な大腸菌宿主の細胞質内において、機能的な組換えscFvを大量に発現させる手法を開発するに至った。
具体的には、特殊な改変を施さない一般的な大腸菌宿主内で、種々のscFv遺伝子を用い、scFvのC末端側にDNA結合タンパク質の１種であるジンクフィンガードメインを3つ有するZif268を融合した「scFv-Zif268」として発現させることによって、組換えscFvの発現量を増大させることができた。得られた組換えscFvは、リーダー配列を含む配列を用いたにもかかわらずペリプラズムではなく細胞質内で発現しており、融合体の状態でも結合活性が高く、ぺリプラズムに発現させたscFvと同程度の結合活性を持っていた。これは、scFv-Zif268が、大腸菌の細胞質内でscFvが正しい立体構造をとった可溶化状態で発現できていることを示している。ペリプラズム発現のためのリーダー配列を省いた追試実験で、全く同じ結果が得られたことからも、「scFv-Zif268」の融合タンパク質が細胞質内で発現し、かつscFvの正常なリフォールディング形成が行われたことが確認できた。この結果は、従来特殊な改変を施さない一般的な大腸菌宿主を用いた場合は、還元的環境下にある細胞質内では機能的なscFvの回収が難しいとされていた技術常識があったことからみて、驚くべき結果である。しかも、Zif268と同様にジンクフィンガードメインに属するGAL4由来DNA結合ドメインを用いた場合には機能的なscFvが取得できない（非特許文献９）ことに加え、ロイシンジッパードメインを用いた場合はペリプラズム内での発現であった（非特許文献１１、１２）ことを考え合わせると、DNA結合ドメインのうちでもZif268もしくはZif268と類似したDNA結合ドメイン特有の効果である可能性がある。
また、その際Zif268をscFvのN端に融合させた「Zif268-scFv」の場合は、発現が確認できなかったという結果を得たことから、両者の結合順序も重要であり、Zif268を機能的な状態で取得したい標的タンパク質scFvのC末側に結合する必要があるといえる。
本発明者らは、さらにZif268と類似のDNA結合ドメインとして、Zif268と同程度の分子量を持ち、2次構造としてαヘリックスを多く含んでいるDNA結合ドメインである、トリプトファン・リプレッサー（trpR）及びHinリコンビナーゼ由来のHinRを選択し、両者に対して「scFv-Zif268」と同様の手法を適用し、大腸菌でscFvのC末端側に融合させた融合タンパク質を発現させたところ、Zif268と同様、細胞質内での機能的なscFvの発現増強作用が確認できた。また、scFvの由来生物種や抗原特異性を変えても同様の結果が得られることも確認できた。
このことから、scFvに対してZif268、trpR又はHinRという特定のDNA結合ドメインを含むタンパク質をC末側に結合させることで、scFvを細胞質内で機能的でかつ可溶性の状態で大量に発現させることができることが判明した。
The present inventors diligently worked on solving this problem, and developed a method for expressing a large amount of functional recombinant scFv in the cytoplasm of a general E. coli host without specially improving the host E. coli. It came to develop.
Specifically, Zif268 having three zinc finger domains, which are one type of DNA-binding protein, is fused to the C-terminal side of scFv using a variety of scFv genes in a general E. coli host without special modification. By expressing as “scFv-Zif268”, the expression level of the recombinant scFv could be increased. The obtained recombinant scFv is expressed not in the periplasm but in the cytoplasm despite the use of the sequence including the leader sequence, and has high binding activity even in the fusion state, and is the same as the scFv expressed in the periplasm. It had a degree of binding activity. This indicates that scFv-Zif268 can be expressed in a solubilized state in which the scFv takes the correct three-dimensional structure in the cytoplasm of E. coli. In a follow-up experiment without the leader sequence for periplasmic expression, the same results were obtained, so that the fusion protein of “scFv-Zif268” was expressed in the cytoplasm and normal refolding of scFv was performed. I was able to confirm. This result is based on the common knowledge that it was difficult to recover functional scFvs in the cytoplasm in a reducing environment when using a general E. coli host without special modification. This is a surprising result. Moreover, in the same manner as Zif268, when a GAL4-derived DNA binding domain belonging to a zinc finger domain is used, a functional scFv cannot be obtained (Non-patent Document 9). In addition, when a leucine zipper domain is used, it is within the periplasm. (Non-Patent Documents 11 and 12), there is a possibility that this is an effect peculiar to a DNA binding domain similar to Zif268 or Zif268 among DNA binding domains.
In addition, in the case of “Zif268-scFv”, in which Zif268 was fused to the N-terminus of scFv, we obtained a result that the expression could not be confirmed. It can be said that it is necessary to bind to the C-terminal side of the target protein scFv that is desired to be obtained.
The present inventors further have a tryptophan repressor (trpR), which is a DNA-binding domain similar to Zif268, a DNA-binding domain having a molecular weight similar to that of Zif268 and containing many α-helices as a secondary structure. When HinR derived from Hin recombinase was selected and the same method as `` scFv-Zif268 '' was applied to both, the fusion protein fused to the C-terminal side of scFv was expressed in E. coli. The functional scFv expression enhancing action was confirmed. It was also confirmed that similar results could be obtained even when the scFv-derived species and antigen specificity were changed.
From this, scFv can be expressed in large quantities in a functional and soluble state in the cytoplasm by binding a protein containing a specific DNA binding domain such as Zif268, trpR or HinR to scFv on the C-terminal side. Turned out to be possible.
次いで、本発明者らは、scFv以外の外来タンパク質であって、従来から形質転換細菌内での正しい立体構造の可溶化状態で発現することが難しいとされていた節足動物由来のガウシア・ルシフェラーゼ（GLuc）、や膜タンパク質のCD8を、scFvと同様にZif268との融合タンパク質として大腸菌内で発現させたところ、正しい立体構造をとった状態で細胞質内で大量に発現させることに成功した。このことは、発現させる対象のタンパク質については、ヒトなど哺乳動物由来のタンパク質でなくても、また本来可溶性のタンパク質ではない膜タンパク質であっても、細菌細胞質内で正しい立体構造をとり、機能的でしかも可溶性の状態で発現させることが可能であることを意味する。
以上の知見を得たことで、本願発明を完成させた。
Next, the present inventors have introduced a Gaussia luciferase derived from an arthropod that is a foreign protein other than scFv and has conventionally been difficult to express in a solubilized state of a correct three-dimensional structure in a transformed bacterium. (GLuc) and the membrane protein CD8 were expressed in Escherichia coli as a fusion protein with Zif268 in the same manner as scFv, and they were successfully expressed in large quantities in the cytoplasm in the correct three-dimensional structure. This means that the target protein to be expressed has a correct three-dimensional structure in the bacterial cytoplasm, whether it is a protein derived from a mammal such as a human or a membrane protein that is not inherently soluble, and is functional. It means that it can be expressed in a soluble state.
By obtaining the above knowledge, the present invention was completed.
すなわち、本願発明は以下の通りである。
〔１〕 所望の外来タンパク質のC末側に、Zif268、trpR、及びHinRから選択されたDNA結合ドメイン又はその機能的フラグメントを含むDNA結合タンパク質を融合させた融合タンパク質をコードする核酸を含む発現ベクターを用いて細菌宿主を形質転換することを特徴とする、細菌宿主細胞質内での機能的外来タンパク質の発現増強方法。
〔２〕 前記融合タンパク質において、所望の外来タンパク質のC末側であって、かつDNA結合タンパク質のN末側の位置に切断可能部位を含むリンカーが設けられていることを特徴とする、前記〔１〕に記載の発現増強方法。
〔３〕 所望の外来タンパク質が、単鎖抗体（scFv）である、前記〔１〕又は〔２〕に記載の発現増強方法。
〔４〕 所望の外来タンパク質のC末側に、Zif268、trpR、及びHinRから選択されたDNA結合ドメイン又はその機能的フラグメントを含むDNA結合タンパク質を融合させた融合タンパク質をコードする核酸を有効成分とする、細菌宿主細胞質内での機能的外来タンパク質発現増強剤。
〔５〕 Zif268、trpR、及びHinRから選択されたDNA結合ドメイン又はその機能的フラグメントを含むDNA結合タンパク質をコードする核酸の使用であって、当該DNA結合タンパク質をコードする核酸を、所望の外来タンパク質をコードする核酸の３’末端に融合させることによって、前記外来タンパク質を細菌宿主の細胞質内で機能的外来タンパク質として発現させるための使用。
〔６〕 細菌宿主における所望の外来タンパク質の発現量を増強させる方法であって、下記の（ａ）及び（ｂ）の工程を含む方法；
（ａ）Zif268、trpR、及びHinRから選択されたDNA結合ドメイン又はその機能的フラグメントを含むDNA結合タンパク質をコードする第１の核酸分子を、前記外来タンパク質をコードする第２の核酸分子の3’末端側に融合させて、細菌宿主における発現産物が、前記DNA結合タンパク質が前記外来タンパク質のＣ末側に位置するような融合タンパク質をコードする遺伝子構築物を形成する工程、
（ｂ）前記構築物を、細菌宿主の細胞質内で発現させる工程。
〔７〕 さらに、下記の工程（ｃ）を含む前記〔６〕に記載の方法；
（ｃ）発現させた融合タンパク質を宿主細菌の細胞質画分から回収する工程。
〔８〕 前記第１の核酸分子の5’末端側で、かつ第２の核酸分子の3’末端側の位置に、切断可能部位を含むリンカー配列をコードする核酸分子が融合されていることを特徴とする前記〔６〕又は〔７〕に記載の方法。
That is, the present invention is as follows.
[1] An expression vector comprising a nucleic acid encoding a fusion protein in which a DNA binding protein containing a DNA binding domain selected from Zif268, trpR and HinR or a functional fragment thereof is fused to the C-terminal side of a desired foreign protein A method for enhancing the expression of a functional foreign protein in a bacterial host cytoplasm, which comprises transforming a bacterial host with
[2] The fusion protein is characterized in that a linker containing a cleavable site is provided at a position on the C-terminal side of a desired foreign protein and on the N-terminal side of a DNA-binding protein. [1] The expression enhancing method according to [1].
[3] The method for enhancing expression according to [1] or [2] above, wherein the desired foreign protein is a single chain antibody (scFv).
[4] A nucleic acid encoding a fusion protein in which a DNA-binding protein selected from Zif268, trpR, and HinR or a functional fragment thereof is fused to the C-terminal side of a desired foreign protein is used as an active ingredient. A functional foreign protein expression enhancer in the bacterial host cytoplasm.
[5] Use of a nucleic acid encoding a DNA binding protein comprising a DNA binding domain selected from Zif268, trpR and HinR or a functional fragment thereof, wherein the nucleic acid encoding the DNA binding protein is converted into a desired foreign protein. Use for expressing the foreign protein as a functional foreign protein in the cytoplasm of a bacterial host by fusing to the 3 ′ end of a nucleic acid encoding
[6] A method for enhancing the expression level of a desired foreign protein in a bacterial host, comprising the following steps (a) and (b):
(A) a first nucleic acid molecule encoding a DNA binding protein comprising a DNA binding domain selected from Zif268, trpR, and HinR or a functional fragment thereof, and 3 ′ of the second nucleic acid molecule encoding the foreign protein. Fusing terminally to form an expression product in a bacterial host to form a genetic construct encoding a fusion protein such that the DNA binding protein is located on the C-terminal side of the foreign protein;
(B) expressing the construct in the cytoplasm of a bacterial host.
[7] The method according to [6], further comprising the following step (c);
(C) recovering the expressed fusion protein from the cytoplasmic fraction of the host bacterium.
[8] A nucleic acid molecule encoding a linker sequence containing a cleavable site is fused to the 5 ′ end side of the first nucleic acid molecule and the 3 ′ end side of the second nucleic acid molecule. [6] or [7], wherein the method is characterized.
本発明により、従来一般的な大腸菌など細菌の細胞質内では、正常な立体構造（本来の折りたたみ構造）を保持し、可溶化した状態で発現させることが困難とされていた外来タンパク質を、そのC末端側に各種DNA結合タンパク質を融合させた融合タンパクとして発現させることで、どのような外来タンパク質であっても細胞質内で正常な立体構造を保持した状態で効率よく発現させることができる。そして、従来のペリプラズム領域よりも空間的なゆとりが大きい細胞質内で発現増強効果を発揮できるため、望みの外来タンパク質を正常な立体構造を保持した可溶化状態で大量に製造することが可能となった。
特に、医薬用途などで期待が大きい組換え単鎖抗体（scFv）を、大量生産することができる点は画期的である。
According to the present invention, an exogenous protein, which has conventionally been considered difficult to express in a solubilized state, retains a normal three-dimensional structure (original folding structure) in the cytoplasm of bacteria such as general E. coli. By expressing as a fusion protein in which various DNA-binding proteins are fused on the terminal side, any foreign protein can be efficiently expressed in a state in which a normal three-dimensional structure is maintained in the cytoplasm. In addition, since the expression enhancing effect can be exerted in the cytoplasm having a larger spatial space than the conventional periplasm region, it becomes possible to produce a large amount of a desired foreign protein in a solubilized state retaining a normal three-dimensional structure. It was.
In particular, it is epoch-making in that a recombinant single chain antibody (scFv), which is highly expected for pharmaceutical use, can be mass-produced.
１．本発明で用いられる「DNA結合タンパク質」について
（１）本発明の「DNA結合タンパク質」
本発明で用いられる「DNA結合タンパク質」は、周知のDNA結合ドメインであるZif268、トリプトファン・リプレッサー（trpR）及びHinリコンビナーゼ由来のHinRから選択されたDNA結合ドメイン又はその機能的フラグメントを含むDNA結合タンパク質である。
「Zif268」は、ステロイド／甲状腺ホルモン核受容体スーパーファミリーのメンバーEgr1（Mus musculus early growth response）から取得された、3つのZnフィンガードメインClass-1を有するDNA結合ドメインであり、様々な生物種で配列上の相同性がきわめて高く保存されている。Zif268は通常のZnフィンガードメインClass-1に特有の、3つのDNA結合配列モチーフ（oxCx2-4Cx3ox5ox2Hx3-4Hx2-6）で構成され、この配列モチーフに含まれる２つのヒスチジン残基および２つのシステイン残基が亜鉛イオンと結合している（例えば、Berg et al. (1996) Science 271:1081-1085参照）。なお、Znフィンガーの配列モチーフ「oxCx2-4Cx3ox5ox2Hx3-4Hx2-6」中、C=Cys、H=His、o=疎水性アミノ酸残基、x=任意のアミノ酸残基を表す。
本実施例では、マカクザル由来のZif268（AF385078(nlm.nih.gov/nuccore/AF385078)）からその機能的フラグメントに当たる19〜107番目の部分アミノ酸配列（配列番号１）を大腸菌に最適化し人工合成して用いたが、マカクザル由来配列には限られず、どのような生物種由来であってもよい。たとえば、ヒトのZif268（NM_001964）の他、mouse（NM_007913.5）、rat（NM_012551.2）、cow（NM_001045875.1）、chicken（NM_204136.2）、bird（AF492528）などが例示できる。これらの全長配列を用いてもいいし、これら配列中の本発明の実施例で用いられた配列番号１に対応する機能的フラグメントを含む部分配列を用いても良い。なお、実施例などでは、当該配列番号１のZif268機能的フラグメントを、単に「Zif268」と表記し、融合タンパク質でも「タンパク質-Zif268」などと表記している。
「HinR」は、Hinリコンビナーゼ（198amino acids）中のC端の52アミノ酸がDNA結合能を有するDNA結合ドメイン（HinR）に相当し、５つのαへリックスで構成されたヘリックス−ターン−ヘリックス（HTH）ドメインに属する（非特許文献１８）。
「HinR」も、各種微生物のインベルターゼ中に保存性の高い領域が確認されており、本実施例では非特許文献１８記載の配列番号３に示されるアミノ酸配列（サルモネラ菌由来）を用いたが、サルモネラ菌由来インベルターゼ（WP_000190914）の他、大腸菌（WP_021553495）、枯草菌（WP_016191666）、肺炎桿菌（WP_020804545）などの配列中の配列番号３に相当するアミノ酸配列を含む機能的フラグメントを利用することができる。
「trpR」は、Tryptophan転写抑制因子であって、「HinR」と同様にHTHドメインに属し、108アミノ酸からなり、トリプトファンの結合に伴い2量体化し、DNAに結合する性質をもち（非特許文献１９）、配列番号５などで表される。
実施例では非特許文献１９に記載の大腸菌由来trpR（PRF Ac.070329A）を用いたが、他の大腸菌（WP_021544456）やサルモネラ菌（WP_000190914）など他の細菌由来の「trpR」を用いることもできる。
1. “DNA-binding protein” used in the present invention (1) “DNA-binding protein” of the present invention
The “DNA binding protein” used in the present invention is a DNA binding domain comprising a DNA binding domain selected from Zif268, tryptophan repressor (trpR) and HinR derived from Hin recombinase, or a functional fragment thereof. It is a protein.
“Zif268” is a DNA-binding domain with three Zn finger domains, Class-1, obtained from Egr1 (Mus musculus early growth response), a member of the steroid / thyroid hormone nuclear receptor superfamily. The sequence homology is very high and conserved. Zif268 is composed of three DNA binding sequence motifs (oxCx 2-4 Cx 3 ox 5 ox 2 Hx 3-4 Hx 2-6 ) unique to the normal Zn finger domain Class-1 and is included in this sequence motif Two histidine residues and two cysteine residues are bound to the zinc ion (see, eg, Berg et al. (1996) Science 271: 1081-1085). In addition, in the sequence motif “oxCx 2-4 Cx 3 ox 5 ox 2 Hx 3-4 Hx 2-6 ” of Zn finger, C = Cys, H = His, o = hydrophobic amino acid residue, x = any amino acid Represents a residue.
In this example, Zif268 (AF385078 (nlm.nih.gov/nuccore/AF385078)) derived from macaque monkey was used to artificially synthesize the 19th to 107th partial amino acid sequence (SEQ ID NO: 1) corresponding to the functional fragment thereof in E. coli. However, the sequence is not limited to the macaque monkey-derived sequence, and may be derived from any species. Examples include human Zif268 (NM_001964), mouse (NM_007913.5), rat (NM_012551.2), cow (NM_001045875.1), chicken (NM_204136.2), bird (AF492528), and the like. These full-length sequences may be used, or a partial sequence containing a functional fragment corresponding to SEQ ID NO: 1 used in Examples of the present invention in these sequences may be used. In Examples and the like, the functional fragment of SEQ ID NO: 1 is simply referred to as “Zif268”, and the fusion protein is also referred to as “protein-Zif268” or the like.
“HinR” corresponds to a DNA-binding domain (HinR) in which 52 C-terminal amino acids in Hin recombinase (198 amino acids) have DNA-binding ability, and a helix-turn-helix (HTH) composed of five α helices. ) Belongs to a domain (Non-patent Document 18).
As for “HinR”, a highly conserved region has been confirmed in the invertase of various microorganisms. In this example, the amino acid sequence (derived from Salmonella) shown in SEQ ID NO: 3 described in Non-Patent Document 18 was used. In addition to the derived invertase (WP_000190914), a functional fragment containing an amino acid sequence corresponding to SEQ ID NO: 3 in the sequence of Escherichia coli (WP_021553495), Bacillus subtilis (WP_016191666), Neisseria pneumoniae (WP_020804545), or the like can be used.
“TrpR” is a Tryptophan transcriptional repressor, which belongs to the HTH domain like “HinR”, consists of 108 amino acids, dimerizes with the binding of tryptophan, and has the property of binding to DNA (non-patent literature). 19), represented by SEQ ID NO: 5 and the like.
In Examples, E. coli-derived trpR (PRF Ac.070329A) described in Non-Patent Document 19 was used, but “trpR” derived from other bacteria such as other E. coli (WP_021544456) and Salmonella (WP_000190914) can also be used.
本発明においては、これら各DNA結合ドメイン（Zif268、HinR又はtrpR）もしくはその機能的フラグメントを含んでさえいれば、それぞれのN端及び／又はC端側に余分なアミノ酸配列が残っていても、又は他のペプチドもしくはタンパク質が結合して融合タンパク質となっても、細菌細胞質内での機能的な外来タンパク質形成能という効果は変わらない。
したがって、本発明の「DNA結合タンパク質」は、以下のように表すことができる。
「Zif268、HinR及びtrpRから選択されたDNA結合ドメイン又はその機能的フラグメントを含むアミノ酸配列からなるDNA結合タンパク質。」
また、これら各DNA結合ドメイン中のαへリックス構造、βシートなどの立体構造に影響を与えない程度のアミノ酸変異、すなわち１ないし数個（10以下、好ましくは5以下、より好ましくは3以下）のアミノ酸の欠失・置換・挿入・付加による変異であれば許容される。
したがって、本発明の各DNA結合ドメインは、それぞれのアミノ酸配列を用いて、以下のように表現することもできる。
「配列番号１、３もしくは５に示されるアミノ酸配列、またはその１ないし数個のアミノ酸が欠失・置換・挿入・付加されたアミノ酸配列であって、DNA結合ドメインとしての活性を保持したアミノ酸配列からなるDNA結合タンパク質。」
In the present invention, as long as each of these DNA binding domains (Zif268, HinR or trpR) or a functional fragment thereof is included, even if an extra amino acid sequence remains on the N-terminal and / or C-terminal side, Alternatively, even if other peptides or proteins are combined to form a fusion protein, the effect of functional foreign protein forming ability in the bacterial cytoplasm remains unchanged.
Therefore, the “DNA binding protein” of the present invention can be expressed as follows.
“A DNA binding protein comprising an amino acid sequence comprising a DNA binding domain selected from Zif268, HinR and trpR or a functional fragment thereof.”
In addition, amino acid mutations that do not affect the three-dimensional structure such as α helix structure and β sheet in each DNA binding domain, that is, 1 to several amino acids (10 or less, preferably 5 or less, more preferably 3 or less) Any mutation caused by deletion, substitution, insertion, or addition of the amino acid is acceptable.
Therefore, each DNA binding domain of the present invention can also be expressed as follows using the respective amino acid sequences.
"Amino acid sequence shown in SEQ ID NO: 1, 3 or 5, or an amino acid sequence in which one to several amino acids are deleted, substituted, inserted, or added, and retaining activity as a DNA binding domain DNA binding protein consisting of. "
（２）本発明で用いる「DNA結合タンパク質」の共通性
本発明の「DNA結合ドメイン」は、いずれも所望の外来タンパク質のC末側に結合させた融合タンパクとして大腸菌など細菌宿主で発現させることで、細菌の細胞質内で、正常なフォールディングを形成させ、かつ発現増強も起こさせる機能を有している。
ところで、DNA結合ドメインは、一般に特定の転写調節機構に関与する転写因子などに由来するため、それぞれ特異的に固有のDNA配列を認識し、当該DNA配列を含む特定のDNA領域への結合能を有しており、従来から、このような個々のDNA結合ドメイン固有の特定DNA領域への結合能を利用して、標的遺伝子の転写制御を行うことは広く行われており、その際に、scFvと標的遺伝子の転写開始領域を認識するDNA結合ドメインとの融合タンパク質の構築も行われていた（特許文献４、５、非特許文献２０）。
本発明の各DNA結合ドメインにもそれぞれ特定のDNA領域に対する特異的な認識能、結合能が存在するが、それぞれの認識配列には全く共通性がないことからみて、このような特定のDNA配列に対する特異的な認識能が利用されているわけでないことは明らかである。具体的には、Zif268の認識配列が「gcgtgggcgt」配列であり、HinRの認識配列が「tttttgataa」配列であり、Tryptophan transcriptional repressor中のtrpRの認識配列が「gtactagtt」配列であって、それぞれのDNA結合ドメインが特異的に認識し、結合する対象となるDNA領域の配列間には、何らの共通性は見いだせない。
(2) Commonality of “DNA-binding protein” used in the present invention The “DNA-binding domain” of the present invention is expressed in a bacterial host such as Escherichia coli as a fusion protein bound to the C-terminal side of a desired foreign protein. In the bacterial cytoplasm, it has a function of forming normal folding and also enhancing expression.
By the way, since DNA binding domains are generally derived from transcription factors involved in specific transcriptional regulatory mechanisms, each specifically recognizes a unique DNA sequence and has the ability to bind to a specific DNA region containing the DNA sequence. Conventionally, transcription of target genes has been widely performed by utilizing such ability to bind to a specific DNA region unique to each DNA binding domain. And a fusion protein of a DNA binding domain that recognizes the transcription initiation region of the target gene have also been constructed (Patent Documents 4 and 5, Non-Patent Document 20).
Each of the DNA binding domains of the present invention also has specific recognition ability and binding ability for a specific DNA region. However, in view of the fact that each recognition sequence has no commonality, such a specific DNA sequence. It is clear that the specific recognition ability for is not exploited. Specifically, the Zif268 recognition sequence is a “gcgtgggcgt” sequence, the HinR recognition sequence is a “tttttgataa” sequence, the trpR recognition sequence in the Tryptophan transcriptional repressor is a “gtactagtt” sequence, and each DNA No commonality can be found between the sequences of DNA regions that are specifically recognized and bound by the binding domain.
本発明のDNA結合ドメインが、融合させた外来タンパク質に対して、どのようなメカニズムで細菌の細胞質内で、正常なフォールディングを形成させ、かつ発現増強も起こさせるのかの詳細は不明であるが、標的とする外来タンパク質のC末側に結合させた、各種のDNA結合ドメインに共通的な性質に基づいて、細胞質内での融合タンパク質中の外来タンパク質の正しいジスルフィド結合形成を促進することができ、その結果、機能的外来タンパク質の産生促進を実現することができたものと解される。本発明者らは、各「DNA結合ドメイン」に共通した性質として、以下のような性質に着目している。すなわち、その１つは、DNA結合ドメイン共通の性質としてマイナス電荷を有するDNAへの結合能を持つことからみて、プラス電荷と共に親水性の傾向を持っているフラグメントであると考えられる点であり、他の１つは、Zif268、HinR及びtrpR中の機能的フラグメント領域を構成するアミノ酸残基数は50〜110程度（約5〜11kDa）とコンパクトであり、しかもαへリックスを多く含む安定かつ柔軟な構造（S. Harrison, Nature, 353: 715-719（1991）のFIG.1など参照）を有している点である。前者の性質は、静電的な相互作用による非特異的なタンパク質、核酸などへの結合能を想起させるものである。以下のメカニズムに限定するものではないが、後者の性質は、シャペロンタンパク質とも共通しており、外来タンパク質との融合タンパク質として発現後、速やかに柔軟構造を利用して外来タンパク質と緩く非特異的な結合状態を形成し、外来タンパク質のリフォールディングを助けるシャペロン機能を発揮するというメカニズムが強く示唆される。 The details of how the DNA-binding domain of the present invention forms a normal folding and enhances expression in the bacterial cytoplasm with respect to the fused foreign protein is unknown, Based on the common properties of various DNA binding domains bound to the C-terminal side of the target foreign protein, it can promote the correct disulfide bond formation of the foreign protein in the fusion protein in the cytoplasm. As a result, it is understood that the production of functional foreign protein can be promoted. The present inventors pay attention to the following properties as properties common to each “DNA binding domain”. That is, one of the points is considered to be a fragment having a hydrophilic tendency with a positive charge in view of the ability to bind to DNA having a negative charge as a common property of the DNA binding domain, The other is a compact and stable amino acid residue consisting of about 50 to 110 (about 5 to 11 kDa) in the functional fragment region in Zif268, HinR, and trpR, and also contains a lot of α helices. (Refer to FIG. 1 of S. Harrison, Nature, 353: 715-719 (1991)). The former property is reminiscent of the ability to bind to non-specific proteins, nucleic acids, etc. by electrostatic interaction. Although not limited to the following mechanism, the latter property is also common to chaperone proteins, and after expression as a fusion protein with a foreign protein, it quickly uses a flexible structure to loosely and nonspecifically bind to the foreign protein. The mechanism that forms a binding state and exerts a chaperone function that helps refolding foreign proteins is strongly suggested.
２．本発明の対象となる外来タンパク質
本発明において対象となる外来タンパク質は、どのようなタンパク質であっても良いが、特に、従来から大腸菌などの細菌の細胞質内では正常な立体構造での発現が難しいとされていたヒトなど哺乳類、鳥類、昆虫など真核生物由来のタンパク質が特に適している。たとえば単鎖抗体などの抗体フラグメント、ルシフェラーゼなどの他、インターフェロン、顆粒球コロニー刺激因子、エリスロポエチン、トロンボポエチン、成長ホルモン、プロインスリンなどの種々の生理活性タンパク質、プロウロキナーゼ、組織プラスミノーゲンアクチベーター、スーパーオキシドディスムターゼなどの酵素類、インターロイキン受容体などのレセプター類、リゾチーム、血清アルブミン、スギ花粉抗原、およびウイルス構成タンパク質などが挙げられる。
抗体フラグメントとしては、単鎖抗体（scFv）の他、Fab又はFvも好ましく用いられる。ここで、単鎖抗体（scFv）は、モノクローナル抗体の重鎖可変領域（VH）と軽鎖可変領域（VL）とをペプチドリンカーでつないだ一本鎖の抗体フラグメントを指し、Fabは、重鎖又は軽鎖由来の可変領域（VH又はVL）と定常領域（CH1又はCL）からなるフラグメント、Fvは重鎖又は軽鎖由来の可変領域（VH又はVL）を指すが、いずれも当業者には周知である。
たとえば、本発明の実施例で用いた「A10B scFv」は、mouse抗ウサギIgG抗体のVHとVLをリンカーで結合した単鎖抗体（AC No.AY635846）であり、典型的なscFvとして研究用に汎用されている。
本発明の単鎖抗体(scFv)として、実施例では、他に由来生物又は抗原が異なるMouse anti-GLuc scFv及びChicken anti-rabbit IgG scFvを用いて同様の結果を得ている。
さらに、本発明の実施例では、DNA結合ドメインと融合させる単鎖抗体以外の機能的外来タンパク質として、実施例では、節足動物由来の「GLuc」及び哺乳動物由来ではあるが膜タンパク質である「CD8」を用いた実験を行い、同様の結果を得ている。なお、「GLuc」は、Gaussia由来ルシフェラーゼ（AY015993）を表し、「CD8」はヒトCD8α（AC No.DQ896639）を表す。
以上のことからみて、DNA結合ドメインと融合させる対象の外来タンパク質は、生物の由来も、タンパク質の種類もどのようなものであっても適用できることは明らかである。
2. Foreign protein which is the subject of the present invention The foreign protein which is the subject of the present invention may be any protein, but in particular, it is difficult to express in a normal three-dimensional structure in the cytoplasm of bacteria such as Escherichia coli. Particularly suitable are proteins derived from eukaryotes such as mammals such as humans, birds and insects. For example, antibody fragments such as single chain antibodies, luciferase, etc., interferon, granulocyte colony stimulating factor, erythropoietin, thrombopoietin, growth hormone, proinsulin and other various physiologically active proteins, prourokinase, tissue plasminogen activator, super Enzymes such as oxide dismutase, receptors such as interleukin receptor, lysozyme, serum albumin, cedar pollen antigen, and virus constituent proteins.
As an antibody fragment, Fab or Fv is preferably used in addition to a single chain antibody (scFv). Here, a single chain antibody (scFv) refers to a single chain antibody fragment in which a heavy chain variable region (V H ) and a light chain variable region (V L ) of a monoclonal antibody are connected by a peptide linker. fragments consisting of the variable regions (V H or V L) and a constant region from the heavy chain or light chain (C H 1 or C L), Fv is the variable region from the heavy chain or light chain (V H or V L) Both are well known to those skilled in the art.
For example, “A10B scFv” used in the examples of the present invention is a single chain antibody (AC No. AY635846) in which V H and V L of mouse anti-rabbit IgG antibody are linked by a linker, and is studied as a typical scFv. It is widely used for.
In the Examples, similar results were obtained using Mouse anti-GLuc scFv and Chicken anti-rabbit IgG scFv having different origins or antigens as the single-chain antibody (scFv) of the present invention.
Furthermore, in the examples of the present invention, as a functional foreign protein other than a single chain antibody to be fused with a DNA binding domain, in the examples, “GLuc” derived from an arthropod and a membrane protein derived from a mammal “ Experiments using CD8 were conducted and similar results were obtained. “GLuc” represents Gaussia-derived luciferase (AY015993), and “CD8” represents human CD8α (AC No. DQ896639).
From the above, it is clear that the target foreign protein to be fused with the DNA binding domain can be applied regardless of the origin of the organism and the type of protein.
３．融合タンパク質及びその製造方法
（１）リンカー、遺伝子の結合順序
本発明では正常な機能的タンパク質として発現させたい所望の外来タンパク質のC末側にDNA結合タンパク質が結合した状態の融合タンパク質が発現できるという融合タンパク質での結合順序が重要である。したがって、発現ベクターに挿入する組換えDNAは、外来タンパク質遺伝子の3’末端側にリンカーを介して又は介することなくDNA結合タンパク質遺伝子を結合させて構築する。
また、本発明では、ペリプラズムなど各器官への移行シグナル配列の有無に関わりなく同様の細胞質内での発現増強効果を発揮させることができる。すなわち、市販の移行シグナル配列、分泌シグナル配列などが組み込まれたベクターなどを用いる場合に、当該シグナル配列をわざわざ除去する必要はないし、反対にあえて結合させる必要はない。ただし、ペリプラズムもしくは細胞外への分泌を完全に抑えるためには、ペリプラズム移行シグナル配列、細胞外分泌シグナル配列などの移行シグナルはあらかじめ全て除去しておくことが好ましい。
ここで、リンカーとしては、汎用のリンカー配列であるグリシン（G）及び／又はセリン（S）の連続配列（たとえば(G4S)3、(G4S)4）を用いることができるが、各種リンカー配列が周知であって、当業者は適宜選定できるので、これには限定されない。なお、前述のように、外来タンパク質遺伝子とDNA結合ドメインとの結合方法自体は従来から既知であり（非特許文献２０、特許文献４、５）、これら文献の記載に従って結合させることができる。
3. Fusion protein and production method thereof (1) Linker, gene binding order In the present invention, a fusion protein in which a DNA binding protein is bound to the C-terminal side of a desired foreign protein to be expressed as a normal functional protein can be expressed. The order of binding in the fusion protein is important. Therefore, the recombinant DNA to be inserted into the expression vector is constructed by binding the DNA-binding protein gene to the 3 ′ end side of the foreign protein gene via or without a linker.
Furthermore, in the present invention, the same cytoplasmic expression enhancing effect can be exhibited regardless of the presence or absence of a signal translocation signal to each organ such as periplasm. That is, when a commercially available vector incorporating a transition signal sequence, a secretory signal sequence, or the like is used, it is not necessary to remove the signal sequence. However, in order to completely suppress periplasm or extracellular secretion, it is preferable to remove all transition signals such as periplasmic transition signal sequences and extracellular secretory signal sequences in advance.
Here, as the linker, a continuous sequence of glycine (G) and / or serine (S) which are general-purpose linker sequences (for example, (G 4 S) 3 , (G 4 S) 4 ) can be used. Various linker sequences are well known and can be appropriately selected by those skilled in the art, and are not limited thereto. As described above, the method of binding a foreign protein gene and a DNA binding domain itself has been conventionally known (Non-patent Document 20, Patent Documents 4 and 5), and can be bound according to the description of these documents.
（２）発現ベクター、宿主微生物
本発明の発現ベクターとしては、通常の細菌用のベクターであれば、どのようなベクターであっても用いることができる。このようなベクターは当業者には周知であり、多数の細菌用ベクターが市販されている。具体的には、例えば、pTrcHis2ベクター、pcDNA3.1/myc-Hisベクター（Invitrogen社製）、pUC119（宝酒造社製）、pBR322（宝酒造社製）、pBluescript II KS+ Stratagene社製）、pQE-Tri（Qiagen社製）、pET、pGEM-3Z、pGEX、pMAL等のプラスミドベクター、λEMBL３（Stratagene社製）、λDASHII（フナコシ社製）等のバクテリオファージベクター、Charomid DNA（和光純薬工業(株)製）、Lorist6（和光純薬工業(株)製）等のコスミドベクター等が挙げられる。また、大腸菌由来のプラスミド(例えばpTrc99A, pKK223, pET3a)等の他、pA1−11、pXT1、pRc/CMV、pRc/RSV、pcDNA I/Neo、p3×FLAG-CMV-14、pCAT3、pcDNA3.1、pCMV等も挙げられる。
ただし、本発明においては、ペリプラズムもしくは、菌体外に分泌させずに、細胞質内で発現させることを特徴としているため、市販のプラスミド、ファージベクターのうち、あらかじめOmpA、pelBなどのシグナルペプチドをコードするDNAが組み込まれているベクターをあえて用いる必要はない。そのようなベクターを用いる場合であって、完全にペリプラズムや菌体外への漏れを防ぐためには、これらシグナル配列を除去又は不活性化して用いる。
また、外来タンパク質の検出や精製を容易にするために、外来タンパク質は、タグペプチドを結合して発現させても良い。融合させるタグペプチドとしてはFLAGタグ、3XFLAGタグ、Hisタグ（His tag、例えば６×Hisタグ）等が挙げられる。
そして、本発明の融合タンパク質遺伝子の転写を制御するためのプロモーターは、誘導可能なプロモーターが好ましく、たとえば、IPTGにより誘導可能なプロモーターであるlac、tac、trcの他、IAAで誘導可能なtrp、L-アラビノースで誘導可能なara、テトラサイクリンを用いて誘導可能なPzt-1、高温（42℃）で誘導可能なPLプロモーター、コールドショック遺伝子の一つであるcspA遺伝子のプロモーターなどが使用できる。これらのプロモーターの制御下にある本発明の融合タンパク質遺伝子を、前記発現ベクターに挿入する。
(2) Expression vector and host microorganism As the expression vector of the present invention, any vector can be used as long as it is a normal vector for bacteria. Such vectors are well known to those skilled in the art and many bacterial vectors are commercially available. Specifically, for example, pTrcHis2 vector, pcDNA3.1 / myc-His vector (Invitrogen), pUC119 (Takara Shuzo), pBR322 (Takara Shuzo), pBluescript II KS + Stratagene), pQE-Tri ( Qiagen), pET, pGEM-3Z, pGEX, pMAL and other plasmid vectors, λEMBL3 (Stratagene), λDASHII (Funakoshi) and other bacteriophage vectors, Charomid DNA (Wako Pure Chemical Industries, Ltd.) And cosmid vectors such as Lorist6 (manufactured by Wako Pure Chemical Industries, Ltd.). In addition to plasmids derived from E. coli (e.g., pTrc99A, pKK223, pET3a), pA1-11, pXT1, pRc / CMV, pRc / RSV, pcDNA I / Neo, p3 × FLAG-CMV-14, pCAT3, pcDNA3.1 And pCMV.
However, in the present invention, since it is characterized in that it is expressed in the cytoplasm without being secreted to the periplasm or outside the cell body, among commercially available plasmids and phage vectors, a signal peptide such as OmpA or pelB is encoded in advance. It is not necessary to use a vector in which the DNA to be integrated is used. In the case of using such a vector, these signal sequences are removed or inactivated in order to completely prevent periplasm or leakage outside the cells.
In order to facilitate the detection and purification of the foreign protein, the foreign protein may be expressed by binding a tag peptide. Examples of the tag peptide to be fused include a FLAG tag, 3XFLAG tag, His tag (His tag, for example, 6 × His tag) and the like.
The promoter for controlling transcription of the fusion protein gene of the present invention is preferably an inducible promoter.For example, in addition to lac, tac, trc which are promoters inducible by IPTG, trp inducible by IAA, derivable L- arabinose ara, Pzt-1 inducible with tetracycline, P L promoter inducible at high temperature (42 ° C.), such as a promoter of cspA gene, one of the cold-shock genes can be used. The fusion protein gene of the present invention under the control of these promoters is inserted into the expression vector.
得られた発現用組み換えベクターを用いて、適当な宿主細菌（原核微生物）を形質転換（形質導入）することにより、形質転換細菌を調製する。
本発明に用いられる宿主細菌としては、大腸菌（Escherichia coli.）の他、バチルス属菌（B. subtilis, B. megaterium, B. brevis, B. borstelenis等）、ブドウ球菌（Staphylococcus）、乳酸連鎖球菌（Lactococcus lactis）、乳酸桿菌（Lactobacillus）などの周知の形質転換用の細菌宿主を用いることができる。
本発明に用いられる宿主大腸菌としては、具体的には、HB101、JM109、MC4100、MG1655、W3110等の一般的に用いられる株を用いることができる。本発明においては、細胞質内の環境を酸化的な環境などの、ジスルフィド結合を形成しやすい細胞質内環境の特別な改良は不要であるが、このような改良が施された市販のコンピテントセル（New England Biolabs社製「SHuffle」又はNovagen社製「Origami」）の他、degP変異株、ompT変異株、tsp変異株、lon変異株、clpPX変異株、hslV/U変異株、lonおよびclpPX二重変異株、lon、clpPXおよびhslV/U三重変異株等のプロテアーゼ変異株、plsX変異株、rpoH欠失変異株、rpoHミスセンス変異株等の変異株を用いることもできる。
A transformed bacterium is prepared by transforming (transducing) an appropriate host bacterium (prokaryotic microorganism) using the obtained recombinant vector for expression.
Examples of the host bacterium used in the present invention include Escherichia coli. Bacillus (B. subtilis, B. megaterium, B. brevis, B. borstelenis, etc.), Staphylococcus, lactic streptococci Well-known bacterial hosts for transformation such as Lactococcus lactis and Lactobacillus can be used.
As the host E. coli used in the present invention, specifically, commonly used strains such as HB101, JM109, MC4100, MG1655, W3110 can be used. In the present invention, there is no need to specially improve the cytoplasmic environment in which disulfide bonds are easily formed, such as an oxidative environment in the cytoplasmic environment. New England Biolabs "SHuffle" or Novagen "Origami"), degP mutant, ompT mutant, tsp mutant, lon mutant, clpPX mutant, hslV / U mutant, lon and clpPX double Mutants such as mutants, protease mutants such as lon, clpPX and hslV / U triple mutants, plsX mutants, rpoH deletion mutants, and rpoH missense mutants can also be used.
発現用組み換えベクターによる宿主細胞の形質転換は、従来周知の方法を用いて行うことができる。例えば、一般的なコンピテントセル形質転換方法（J. Bacteriol. 93, 1925 (1967)）、プロトプラスト形質転換法（Mol. Gen. Genet. 168, 111 (1979)）、エレクトロポレーション法（FEMS Microbiol. Lett. 55, 135 (1990)）又はLP形質転換方法（T. Akamatsuら, Bioscience, Biotechnology, and Biochemistry, 2001, 65, 4, p.823-829）、M.Morrisonの方法（Methods in Enzymology, 68, 326-331,1979参照）等により行うことができる。また、市販のコンピテントセルを用いる場合には、その製品プロトコールに従って形質転換を行えばよい。 Transformation of a host cell with a recombinant expression vector can be performed using a conventionally well-known method. For example, a general competent cell transformation method (J. Bacteriol. 93, 1925 (1967)), a protoplast transformation method (Mol. Gen. Genet. 168, 111 (1979)), an electroporation method (FEMS Microbiol). Lett. 55, 135 (1990)) or LP transformation method (T. Akamatsu et al., Bioscience, Biotechnology, and Biochemistry, 2001, 65, 4, p. 823-829), M. Morrison method (Methods in Enzymology 68, 326-331, 1979). In addition, when a commercially available competent cell is used, transformation may be performed according to the product protocol.
（３）外来タンパク質の回収方法、精製方法
形質転換細菌の培養方法は、周知の一般的な細菌の培養方法が適用でき、温度、培地のpH及び培養時間も適宜設定され得る。宿主細胞が大腸菌である形質転換体を培養する場合は、大腸菌を培養する常法の条件で、かつ通常用いられる液体培地で行えばよい。
外来タンパク質は、DNA結合ドメインの融合タンパク質として、培養物から回収される。濾過または遠心分離などの方法によって菌体を回収し、適当な緩衝液に再懸濁する。そして、例えば界面活性剤処理、超音波処理、リゾチーム処理、凍結融解などの方法で、回収された菌体の細胞壁及び／又は細胞膜を破壊した後、遠心分離や濾過などの方法で融合タンパク質を含有する粗抽出液を得る。そして、一般に用いられる方法に従って、粗抽出液から融合タンパク質を単離、精製する。
融合タンパク質の単離、精製方法としては、例えば塩析、溶媒沈殿法等の溶解度を利用する方法、透析、限外濾過、ゲル濾過、ドデシル硫酸ナトリウム−ポリアクリルアミドゲル電気泳動など分子量の差を利用する方法、イオン交換クロマトグラフィーなどの荷電を利用する方法、アフィニティークロマトグラフィーなどの特異的親和性を利用する方法、逆相高速液体クロマトグラフィーなどの疎水性の差を利用する方法、等電点電気泳動などの等電点の差を利用する方法などが挙げられる。精製された融合タンパク質は、例えば抗His抗体を用いたELISA等により確認することができる。
次いで、Enterokinase、Factor Xa、Thrombin、HRV 3C protease、等のタンパク質切断酵素を利用することで、市販ベクターに組み込まれているタグ配列を適宜取り除くことができる。
また、融合タンパク質の構築に当たり、あらかじめ外来タンパク質とDNA結合タンパク質との間のリンカー部分にこれらの酵素の認識領域を組み込んでおくことで、上記切断酵素を利用して、融合タンパク質から外来タンパク質を切断できるから、必要に応じて、外来タンパク質を、外来タンパク質に応じた公知の精製方法に従って単離・精製することができる。
例えば、塩析、イオン交換クロマトグラフィー、疎水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、ゲル濾過クロマトグラフィーに代表されるタンパク質の精製方法により行なうことができる。
(3) Foreign protein recovery method and purification method As a method for culturing transformed bacteria, a well-known general method for culturing bacteria can be applied, and the temperature, the pH of the medium, and the culture time can be appropriately set. When cultivating a transformant whose host cell is Escherichia coli, it may be carried out under the usual conditions for culturing Escherichia coli and in a liquid medium usually used.
The foreign protein is recovered from the culture as a fusion protein of the DNA binding domain. The cells are collected by a method such as filtration or centrifugation and resuspended in an appropriate buffer. For example, after disrupting the cell wall and / or cell membrane of the collected cells by a method such as surfactant treatment, ultrasonic treatment, lysozyme treatment, freeze thawing, etc., the fusion protein is contained by a method such as centrifugation or filtration. To obtain a crude extract. Then, according to a generally used method, the fusion protein is isolated and purified from the crude extract.
Fusion protein isolation / purification methods include, for example, methods using solubility such as salting out and solvent precipitation, dialysis, ultrafiltration, gel filtration, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Method using charge such as ion exchange chromatography, method utilizing specific affinity such as affinity chromatography, method utilizing difference in hydrophobicity such as reversed-phase high performance liquid chromatography, isoelectric point electricity For example, a method using the difference in isoelectric point such as electrophoresis. The purified fusion protein can be confirmed by, for example, ELISA using an anti-His antibody.
Subsequently, by using a protein cleaving enzyme such as Enterokinase, Factor Xa, Thrombin, HRV 3C protease, etc., the tag sequence incorporated in the commercially available vector can be appropriately removed.
In addition, when constructing a fusion protein, the recognition region of these enzymes is incorporated into the linker portion between the foreign protein and the DNA-binding protein in advance, so that the foreign protein is cleaved from the fusion protein using the above-mentioned cleavage enzyme. Therefore, if necessary, the foreign protein can be isolated and purified according to a known purification method according to the foreign protein.
For example, it can be performed by protein purification methods such as salting out, ion exchange chromatography, hydrophobic chromatography, affinity chromatography, and gel filtration chromatography.
以下、実施例により本発明をより具体的に説明するが、本発明はこれら実施例により何ら限定されるものではない。
本発明におけるその他の用語や概念は、当該分野において慣用的に使用される用語の意味に基づくものであり、本発明を実施するために使用する様々な技術は、特にその出典を明示した技術を除いては、公知の文献等に基づいて当業者であれば容易かつ確実に実施可能である。また、各種の分析などは、使用した分析機器又は試薬、キットの取り扱い説明書、カタログなどに記載の方法を準用して行った。
なお、本明細書中に引用した技術文献、特許公報及び特許出願明細書中の記載内容は、本発明の記載内容として参照されるものとする。
EXAMPLES Hereinafter, although an Example demonstrates this invention more concretely, this invention is not limited at all by these Examples.
Other terms and concepts in the present invention are based on the meanings of terms that are conventionally used in the field, and various techniques used to implement the present invention include those that clearly indicate the source. Except for this, it can be easily and reliably carried out by those skilled in the art based on known documents and the like. In addition, various analyzes were performed by applying the methods described in the analytical instruments or reagents used, kit instruction manuals, catalogs, and the like.
In addition, the description content in the technical literature, the patent publication, and the patent application specification cited in this specification shall be referred to as the description content of the present invention.
（実施例１）発現ベクターの作製
（１−１）pET-22b/A10B scFvの作製
A10Bを大腸菌で発現させるための発現ベクターを作製した。AC No.AY635846の配列情報を元に人工合成したA10B遺伝子（配列番号７）の5’末端側にEcoRI、3’末端側にHindIIIとNotIを配置した。当該末端処理を施したA10B遺伝子及び、pET-22b(+)（タカラバイオ社）をEcoRI（Takara社）とNotI（Takara社）で37℃、3時間処理し、アガロースゲル電気泳動後、各バンドを回収しQiaquick Gel Extraction Kit（Qiagen社）を用いて精製した。精製したA10B遺伝子とpET-22b(+)ベクターをライゲーションし、大腸菌（DH5α株）にトランスフォーメーションし、クローニングを行なった。クローンは配列解析を行い、正しい配列のクローンを決定し、pET-22b/A10Bプラスミドを取得した。構築した発現ベクターの模式図を図１に示す。
(Example 1) Preparation of expression vector (1-1) Preparation of pET-22b / A10B scFv
An expression vector for expressing A10B in E. coli was prepared. EcoRI was placed on the 5 ′ end side and HindIII and NotI were placed on the 3 ′ end side of the artificially synthesized A10B gene (SEQ ID NO: 7) based on the sequence information of AC No. AY635846. The end-treated A10B gene and pET-22b (+) (Takara Bio) were treated with EcoRI (Takara) and NotI (Takara) at 37 ° C for 3 hours, and each band was subjected to agarose gel electrophoresis. Was collected and purified using Qiaquick Gel Extraction Kit (Qiagen). The purified A10B gene and pET-22b (+) vector were ligated, transformed into E. coli (DH5α strain), and cloned. The clone was subjected to sequence analysis to determine a clone having the correct sequence, and the pET-22b / A10B plasmid was obtained. A schematic diagram of the constructed expression vector is shown in FIG.
（１−２）pET-22b/A10B scFv-Zif268の作製
A10B scFv-Zif268を大腸菌で発現させるための発現ベクターを作製した。Zif268（AC No.AF385078）中の機能的フラグメント（配列番号１）の配列情報を元にZif268遺伝子断片（配列番号２）を人工合成した。人工的に合成したZif268遺伝子断片が挿入されたプラスミド及び前記（１−１）で作製したpET-22b/A10B scFvをHindIII（Takara社）とXhoI（Takara社）で37℃、3時間処理し、アガロースゲル電気泳動後、各バンドを回収しQiaquick Gel Extraction Kit（Qiagen社）を用いて精製した。精製したZif268遺伝子断片（以下、単にZif268遺伝子という。）とA10B scFvが付加されたpET-22bベクターをライゲーションし、大腸菌にトランスフォーメーションし、クローニングを行なった。クローンは配列解析を行い、正しい配列のクローンを決定し、pET-22b/A10B scFv-Zif268プラスミドを取得した。Zif268はA10B scFvの3’末端側（C末端側）に配置した。構築した発現ベクターの模式図を図１に示す。
(1-2) Preparation of pET-22b / A10B scFv-Zif268
An expression vector for expressing A10B scFv-Zif268 in E. coli was prepared. The Zif268 gene fragment (SEQ ID NO: 2) was artificially synthesized based on the sequence information of the functional fragment (SEQ ID NO: 1) in Zif268 (AC No. AF385078). The plasmid in which the artificially synthesized Zif268 gene fragment was inserted and the pET-22b / A10B scFv prepared in (1-1) above were treated with HindIII (Takara) and XhoI (Takara) at 37 ° C. for 3 hours, After agarose gel electrophoresis, each band was collected and purified using Qiaquick Gel Extraction Kit (Qiagen). The purified Zif268 gene fragment (hereinafter simply referred to as Zif268 gene) and the pET-22b vector to which A10B scFv was added were ligated, transformed into E. coli, and cloned. The clone was subjected to sequence analysis to determine a clone having the correct sequence, and the pET-22b / A10B scFv-Zif268 plasmid was obtained. Zif268 was arranged on the 3 ′ end side (C terminal side) of A10B scFv. A schematic diagram of the constructed expression vector is shown in FIG.
（１−３）pET-30a/A10B scFv-Zif268の作製
A10B scFv-Zif268を大腸菌の細胞質内で発現させるための発現ベクターを作製した。前記（１−２）で作製したpET-22b/A10B scFv-Zif268を鋳型に5’側にNcoIサイトを付加したフォワードプライマー（5’ ATGCCCATGGGTATGGCCCAGGTGCAGCT 3’：配列番号８）及び5’側にXhoIサイトを付加したリバースプライマー（5’ ACGCGCTCGAGGTCTTTCTGGCGTAAG 3’：配列番号９）、を用いてPCR反応によりA10B scFv-Zif268遺伝子を増幅した。PCRはKOD-FXポリメラーゼ（ToYoBo社）を用いて95℃ 2分を1サイクル、95℃ 30秒・55℃ 30秒・68℃ 1分を25サイクル、68℃ 3分・20℃を1サイクルの反応を行った。増幅したA10B scFv-Zif268断片とpET-30aはNcoIとXhoIによりそれぞれ37℃、3時間処理し、アガロースゲル電気泳動後、各バンドを回収しQiaquick Gel Extraction Kitを用いて精製した。精製したA10B scFv-Zif268遺伝子とpET-30aベクターをライゲーションし、大腸菌にトランスフォーメーションし、クローニングを行なった。クローンは配列解析を行い、正しい配列のクローンを決定し、pET-30a/A10B scFv-Zif268プラスミドを取得した。構築した発現ベクターの模式図を図１に示す。
(1-3) Preparation of pET-30a / A10B scFv-Zif268
An expression vector for expressing A10B scFv-Zif268 in the cytoplasm of E. coli was prepared. The forward primer (5 ′ ATGCCCATGGGTATGGCCCAGGTGCAGCT 3 ′: SEQ ID NO: 8) with the NcoI site added to the 5 ′ side using the pET-22b / A10B scFv-Zif268 prepared in (1-2) as a template and the XhoI site on the 5 ′ side A10B scFv-Zif268 gene was amplified by PCR reaction using the added reverse primer (5 ′ ACGCGCTCGAGGTCTTTCTGGCGTAAG 3 ′: SEQ ID NO: 9). PCR uses KOD-FX polymerase (ToYoBo) for 1 cycle at 95 ° C for 2 minutes, 25 cycles at 95 ° C for 30 seconds, 55 ° C for 30 seconds, 68 ° C for 1 minute, and 68 ° C for 3 minutes at 20 ° C for 1 cycle. Reaction was performed. The amplified A10B scFv-Zif268 fragment and pET-30a were each treated with NcoI and XhoI at 37 ° C. for 3 hours. After agarose gel electrophoresis, each band was recovered and purified using the Qiaquick Gel Extraction Kit. The purified A10B scFv-Zif268 gene and the pET-30a vector were ligated, transformed into E. coli, and cloned. The clone was subjected to sequence analysis to determine a clone having the correct sequence, and the pET-30a / A10B scFv-Zif268 plasmid was obtained. A schematic diagram of the constructed expression vector is shown in FIG.
（１−４）pET-22b/A10B scFv-MBPの作製
比較のために、A10B scFvをマルトース結合タンパク質（Maltose-binding protein：MBP）と融合させたA10B scFv-MBPを大腸菌で発現させるための発現ベクターを作製した。
MBPが挿入された市販ベクターのpMAL-p2X（New England Biolabs社）を鋳型に5’側にHindIIIサイトを付加したフォワードプライマー（5’ AGGCAAGCTTAAAATCGAAGAAGGTAAACT 3’：配列番号１０）及び5’にXhoIサイトを付加したリバースプライマー（5’ ACGCGCTCGAGAGTCTGCGCGTCTTTCAGG 3’：配列番号１１）、を用いてPCR反応によりMBP遺伝子を増幅した。PCRはKOD-FXポリメラーゼを用いて95℃ 2分を1サイクル、95℃ 30秒・55℃ 30秒・68℃ 1分を25サイクル、68℃ 3分・20℃を1サイクルの反応を行った。増幅したMBP断片と前記（１−１）で作製したpET-22b/A10B scFvをHindIIIとXhoIで37℃、3時間処理し、アガロースゲル電気泳動後、各バンドを回収しQiaquick Gel Extraction Kitを用いて精製した。精製したMBP遺伝子とA10B scFvが付加されたpET-22bベクターをライゲーションし、大腸菌にトランスフォーメーションし、クローニングを行なった。クローンは配列解析を行い、正しい配列のクローンを決定し、A10BのC末端側にMBPが配置されたpET-22b/A10B scFv-MBPプラスミドを取得した。
(1-4) Preparation of pET-22b / A10B scFv-MBP For comparison, expression to express A10B scFv-MBP fused with maltose-binding protein (MBP) in E. coli A vector was prepared.
Forward primer (5 'AGGCAAGCTTAAAATCGAAGAAGGTAAACT 3': SEQ ID NO: 10) with 5 'added to the 5' side using a commercial vector pMAL-p2X (New England Biolabs) with MBP inserted as a template, and XhoI site added to 5 ' The MBP gene was amplified by PCR reaction using the reverse primer (5 ′ ACGCGCTCGAGAGTCTGCGCGTCTTTCAGG 3 ′: SEQ ID NO: 11). PCR was performed using KOD-FX polymerase for 1 cycle of 95 ° C for 2 minutes, 25 cycles of 95 ° C for 30 seconds, 55 ° C for 30 seconds, 68 ° C for 1 minute, and 68 ° C for 3 minutes at 20 ° C for 1 cycle. . The amplified MBP fragment and pET-22b / A10B scFv prepared in (1-1) above were treated with HindIII and XhoI at 37 ° C for 3 hours, and after agarose gel electrophoresis, each band was recovered and used with the Qiaquick Gel Extraction Kit And purified. The purified MBP gene and pET-22b vector added with A10B scFv were ligated, transformed into E. coli, and cloned. The clone was subjected to sequence analysis to determine a clone having the correct sequence, and a pET-22b / A10B scFv-MBP plasmid in which MBP was placed on the C-terminal side of A10B was obtained.
（１−５）pET-22b/Anti-GLuc scFvの作製
Anti Gaussiaルシフェラーゼ（GLuc）scFvを大腸菌で発現させる発現ベクターを作製した。Anti GLuc scFv（GL-11-6）（配列番号１２）をコードするDNAを鋳型に5’側にEcoRIサイトを付加したフォワードプライマー（5’ ACGCGGAATTCCAGGTGCAGCTGAAGG 3’：配列番号１３）及び5’側にNotIサイトを付加したリバースプライマー（5’ CAAATGCGGCCGCGACGTTTGAGCTCCAG 3’：配列番号１４）、を用いてPCR反応によりAnti-GLuc scFv遺伝子を増幅した。PCRはKOD-FXポリメラーゼを用いて95℃ 2分を1サイクル、95℃ 30秒・55℃ 30秒・68℃ 1分を25サイクル、68℃ 3分・20℃を1サイクルの反応を行った。増幅したAnti GLuc scFv断片とpET-22bをEcoRIとNotIで37℃、3時間処理し、アガロースゲル電気泳動後、各バンドを回収しQiaquick Gel Extraction Kitを用いて精製した。精製したAnti GLuc scFv遺伝子とpET-22bベクターをライゲーションし、大腸菌にトランスフォーメーションし、クローニングを行なった。クローンは配列解析を行い、正しい配列のクローンを決定し、pET-22b/Anti GLuc scFvプラスミドを取得した。
(1-5) Preparation of pET-22b / Anti-GLuc scFv
An expression vector for expressing Anti Gaussia luciferase (GLuc) scFv in E. coli was prepared. Forward primer (5 ′ ACGCGGAATTCCAGGTGCAGCTGAAGG 3 ′: SEQ ID NO: 13) with 5 ′ EcoRI site added to DNA encoding Anti GLuc scFv (GL-11-6) (SEQ ID NO: 12) as template and NotI on 5 ′ side The Anti-GLuc scFv gene was amplified by PCR using the reverse primer (5 ′ CAAATGCGGCCGCGACGTTTGAGCTCCAG 3 ′: SEQ ID NO: 14) to which the site was added. PCR was performed using KOD-FX polymerase for 1 cycle of 95 ° C for 2 minutes, 25 cycles of 95 ° C for 30 seconds, 55 ° C for 30 seconds, 68 ° C for 1 minute, and 68 ° C for 3 minutes at 20 ° C for 1 cycle. . The amplified Anti GLuc scFv fragment and pET-22b were treated with EcoRI and NotI at 37 ° C. for 3 hours. After agarose gel electrophoresis, each band was recovered and purified using the Qiaquick Gel Extraction Kit. The purified Anti GLuc scFv gene and the pET-22b vector were ligated, transformed into E. coli, and cloned. The clone was subjected to sequence analysis to determine a clone having the correct sequence, and the pET-22b / Anti GLuc scFv plasmid was obtained.
（１−６）pET-22b/Anti-GLuc scFv-Zif268の作製
Anti-GLuc scFv-Zif268を大腸菌で発現させるための発現ベクターを作製した。前記（１−２）で作製したpET-22b/A10B scFv-Zif268をEcoRIとNotIで37℃、3時間処理し、アガロースゲル電気泳動後、Zif268が付加されたベクターのバンドを回収しQiaquick Gel Extraction Kitを用いて精製した。精製した遺伝子と前記（１−５）で作製した、EcoRI・NotI処理のAnti-GLuc scFv断片をライゲーションし、大腸菌にトランスフォーメーションし、クローニングを行なった。クローンは配列解析を行い、正しい配列のクローンを決定し、Anti-GLuc scFvのC末端側にZif268が配置されたpET-22b/Anti GLuc scFv-Zif268プラスミドを取得した。
(1-6) Preparation of pET-22b / Anti-GLuc scFv-Zif268
An expression vector for expressing Anti-GLuc scFv-Zif268 in E. coli was prepared. The pET-22b / A10B scFv-Zif268 prepared in (1-2) above was treated with EcoRI and NotI at 37 ° C. for 3 hours. After agarose gel electrophoresis, the vector band to which Zif268 was added was recovered and Qiaquick Gel Extraction Purified using Kit. The purified gene and the EcoRI / NotI-treated Anti-GLuc scFv fragment prepared in (1-5) above were ligated, transformed into Escherichia coli, and cloned. The clone was subjected to sequence analysis to determine a clone having the correct sequence, and a pET-22b / Anti GLuc scFv-Zif268 plasmid in which Zif268 was placed on the C-terminal side of Anti-GLuc scFv was obtained.
（１−７）pET-22b/Chicken anti-rabbit IgG scFv及びpET-22b/Chicken anti-rabbit IgG scFv-Zif268の作製
前記（１−１）と同様の方法で、Chickenの脾臓から取得したChicken anti-rabbit IgG scFv（配列番号１５）をコードするDNAを鋳型としてPCR反応により増幅し、末端をEcoRI・NotI処理したChicken anti-rabbit IgG scFv断片を精製した。当該断片をpET-22bベクターに挿入し、Chicken anti-rabbit IgG scFvを大腸菌内で発現させるための発現ベクター、pET-22b/Chicken anti-rabbit IgG scFvプラスミドを作製した。
前記（１−２）で作製したpET-22b/A10B scFv-Zif268からEcoRI・NotI処理によりZif268が付加されたベクターを回収し、上記EcoRI・NotI処理のChicken anti-rabbit IgG scFv断片をライゲーションし、前記（１−２）と同様の方法により、Anti-rabbit IgG scFvのC末端側にZif268が配置されたpET-22b/Chicken anti-rabbit IgG scFv-Zif268プラスミドを取得した。
(1-7) Production of pET-22b / Chicken anti-rabbit IgG scFv and pET-22b / Chicken anti-rabbit IgG scFv-Zif268 In the same manner as in (1-1) above, chicken anti-chicken obtained from Chicken spleen -Chicken anti-rabbit IgG scFv fragment was amplified by PCR reaction using DNA encoding -rabbit IgG scFv (SEQ ID NO: 15) as a template, and the end was treated with EcoRI / NotI. The fragment was inserted into a pET-22b vector to prepare an expression vector for expressing Chicken anti-rabbit IgG scFv in E. coli, pET-22b / Chicken anti-rabbit IgG scFv plasmid.
Collecting the Zif268-added vector by EcoRI / NotI treatment from the pET-22b / A10B scFv-Zif268 prepared in (1-2) above, ligating the EcoRI / NotI-treated Chicken anti-rabbit IgG scFv fragment, The pET-22b / Chicken anti-rabbit IgG scFv-Zif268 plasmid in which Zif268 was arranged on the C-terminal side of the Anti-rabbit IgG scFv was obtained by the same method as in (1-2) above.
（１−８）pET-22b/A10B scFv-trpRの作製
A10B scFv-trpRを大腸菌で発現させるための発現ベクターを作製した。trpR遺伝子の配列情報（Proc. Natl. Acad. Sci. USA Vol.77, No.12, pp.7117-7121, December 1980）からtrpR遺伝子断片を人工的に合成し（配列番号６）、前記（１−２）と同様に発現ベクターを得た。
(1-8) Preparation of pET-22b / A10B scFv-trpR
An expression vector for expressing A10B scFv-trpR in E. coli was prepared. A trpR gene fragment was artificially synthesized from the sequence information of the trpR gene (Proc. Natl. Acad. Sci. USA Vol. 77, No. 12, pp. 7117-7121, December 1980) (SEQ ID NO: 6). An expression vector was obtained in the same manner as in 1-2).
（１−９）pET-22b/A10B scFv-HinRの作製
A10B-scFv-HinRを大腸菌で発現させるための発現ベクターを作製した。HinRのアミノ酸情報（SCIENCE Vol.263 p.348-355 JANUARY 1994、配列番号３）からコドンを大腸菌に最適化してHinR遺伝子断片（配列番号４）を人工合成し、前記（１−２）と同様に発現ベクターを得た。
(1-9) Preparation of pET-22b / A10B scFv-HinR
An expression vector for expressing A10B-scFv-HinR in E. coli was prepared. The HinR gene fragment (SEQ ID NO: 4) was artificially synthesized from the amino acid information of HinR (SCIENCE Vol.263 p.348-355 JANUARY 1994, SEQ ID NO: 3) by optimizing the codon to Escherichia coli. An expression vector was obtained.
（１−１０）pET-22b/GLucの作製
Gaussiaルシフェラーゼ（GLuc）を大腸菌で発現させる発現ベクターを作製した。Gaussiaルシフェラーゼ遺伝子（AC No.AY015993：配列番号１６）を人工的に合成し、EcoRIとNotIで切断し、切断断片を同様の制限酵素で切断したベクター、pET-22bを前記（１−２）と同様にライゲーション・トランスフォーメーション後、出現したクローンをクローニングし、Gaussiaルシフェラーゼの発現ベクターを構築した。
(1-10) Preparation of pET-22b / GLuc
An expression vector for expressing Gaussia luciferase (GLuc) in E. coli was prepared. A Gaussia luciferase gene (AC No. AY015993: SEQ ID NO: 16) was artificially synthesized, cleaved with EcoRI and NotI, and the cleaved fragment was cleaved with the same restriction enzyme, pET-22b as described in (1-2) above Similarly, after ligation transformation, clones that appeared were cloned, and an expression vector for Gaussia luciferase was constructed.
（１−１１）pET-22b/GLuc-Zif268の作製
GLuc-Zif268を大腸菌で発現させる発現ベクターを作製した。前記（１−２）で作製したpET-22b/A10B scFv-Zif268をEcoRIとNotIで37℃、3時間処理し、アガロースゲル電気泳動後、Zif268が付加されたベクターのバンドを回収しQiaquick Gel Extraction Kitを用いて精製した。精製した遺伝子と前記（１−１０）で作製した、EcoRI・NotI処理のGLuc断片をライゲーションし、大腸菌にトランスフォーメーションし、クローニングを行なった。クローンは配列解析を行い、正しい配列のクローンを決定し、pET-22b/GLuc-Zif268プラスミドを取得した。
(1-11) Preparation of pET-22b / GLuc-Zif268
An expression vector for expressing GLuc-Zif268 in E. coli was prepared. The pET-22b / A10B scFv-Zif268 prepared in (1-2) above was treated with EcoRI and NotI at 37 ° C. for 3 hours. After agarose gel electrophoresis, the vector band to which Zif268 was added was recovered and Qiaquick Gel Extraction Purified using Kit. The purified gene and the EcoRI / NotI-treated GLuc fragment prepared in (1-10) above were ligated, transformed into Escherichia coli, and cloned. The clone was subjected to sequence analysis to determine a clone having the correct sequence, and the pET-22b / GLuc-Zif268 plasmid was obtained.
（１−１２）pET-22b/CD8の作製
CD8（Human CD8α：AC No.DQ896639：配列番号１７）を大腸菌で発現させる発現ベクターを作製した。Human CD8αは遺伝子配列情報を元に人工合成し、合成した遺伝子を鋳型に5’側にEcoRIサイトを付加したフォワードプライマー（5’ -ACGCGGAATTCGTACAAAAGAGCAGGCTC- 3’：配列番号１８）及び5’側にNotIサイトを付加したリバースプライマー（5’ -CCTTTTGCGGCCGCGTACAAGAAAGCTGGGT- 3’：配列番号１９）、を用いてPCR反応によりCD8遺伝子を増幅した。PCRはKOD-FXポリメラーゼを用いて95℃ 2分を1サイクル、95℃ 30秒・55℃ 30秒・68℃ 1分を25サイクル、68℃ 3分・20℃を1サイクルの反応を行った。増幅したCD8断片とpET-22bをEcoRIとNotIで37℃、3時間処理し、アガロースゲル電気泳動後、各バンドを回収しQiaquick Gel Extraction Kitを用いて精製した。精製したCD8遺伝子とpET-22bベクターをライゲーションし、大腸菌にトランスフォーメーションし、クローニングを行なった。クローンは配列解析を行い、正しい配列のクローンを決定し、pET-22b/CD8プラスミドを取得した。
(1-12) Preparation of pET-22b / CD8
An expression vector for expressing CD8 (Human CD8α: AC No. DQ896639: SEQ ID NO: 17) in E. coli was prepared. Human CD8α is artificially synthesized based on gene sequence information, forward primer (5'-ACGCGGAATTCGTACAAAAGAGCAGGCTC-3 ': SEQ ID NO: 18) with 5'-side added EcoRI site using the synthesized gene as template and NotI site on 5'-side The CD8 gene was amplified by PCR reaction using a reverse primer (5′-CCTTTTGCGGCCGCGTACAAGAAAGCTGGGT-3 ′: SEQ ID NO: 19) to which was added. PCR was performed using KOD-FX polymerase for 1 cycle of 95 ° C for 2 minutes, 25 cycles of 95 ° C for 30 seconds, 55 ° C for 30 seconds, 68 ° C for 1 minute, and 68 ° C for 3 minutes at 20 ° C for 1 cycle. . The amplified CD8 fragment and pET-22b were treated with EcoRI and NotI at 37 ° C. for 3 hours, and after agarose gel electrophoresis, each band was recovered and purified using the Qiaquick Gel Extraction Kit. The purified CD8 gene and the pET-22b vector were ligated, transformed into E. coli, and cloned. The clone was subjected to sequence analysis to determine a clone having the correct sequence, and the pET-22b / CD8 plasmid was obtained.
（１−１３）pET-22b/CD8-Zif268の作製
CD8-Zif268を大腸菌で発現させる発現ベクターを作製した。前記（１−２）で作製したpET-22b/A10B scFv-Zif268をEcoRIとNotIで37℃、3時間処理し、アガロースゲル電気泳動後、Zif268が付加されたベクターのバンドを回収しQiaquick Gel Extraction Kitを用いて精製した。精製した遺伝子と前記（１−１２）で作製した、EcoRI・NotI処理のCD8断片をライゲーションし、大腸菌にトランスフォーメーションし、クローニングを行なった。クローンは配列解析を行い、正しい配列のクローンを決定し、pET-22b/CD8-Zif268プラスミドを取得した。
(1-13) Preparation of pET-22b / CD8-Zif268
An expression vector for expressing CD8-Zif268 in E. coli was prepared. The pET-22b / A10B scFv-Zif268 prepared in (1-2) above was treated with EcoRI and NotI at 37 ° C. for 3 hours. After agarose gel electrophoresis, the vector band to which Zif268 was added was recovered and Qiaquick Gel Extraction Purified using Kit. The purified gene and the EcoRI / NotI-treated CD8 fragment prepared in (1-12) above were ligated, transformed into Escherichia coli, and cloned. The clone was subjected to sequence analysis to determine a clone having the correct sequence, and the pET-22b / CD8-Zif268 plasmid was obtained.
（実施例２）Zif268によるA10B scFvの発現増強効果
（２−１）発現ベクターによるタンパク質の発現
実施例１で作製したpET-22b/A10B scFv、pET-22b/A10B scFv-MBP、pET-22b/A10B scFv-Zif268を大腸菌BL21（DE3）株（Novagen社）へ導入し、形質転換クローンをクローニングした。形質転換クローンはアンピシリンを添加したLB培地2mL、37℃、200r.p.mで一晩前培養する。前培養液1mLをアンピシリンを添加したLB培地100mLに加え、37℃、200r.p.mで培養する。波長600nmの吸光度が0.5に達するまで2〜3時間培養し、培養液の温度を30℃に冷し、0.2mMのIPTGを加え、30℃で一晩培養し、各タンパク質の発現を誘導し、大腸菌を回収した。
(Example 2) Expression enhancement effect of A10B scFv by Zif268 (2-1) Expression of protein by expression vector pET-22b / A10B scFv, pET-22b / A10B scFv-MBP, pET-22b / produced in Example 1 A10B scFv-Zif268 was introduced into E. coli BL21 (DE3) strain (Novagen) and the transformed clone was cloned. Transformed clones are pre-cultured overnight in 2 mL of LB medium supplemented with ampicillin at 37 ° C. and 200 rpm. 1 mL of the preculture is added to 100 mL of LB medium supplemented with ampicillin and cultured at 37 ° C. and 200 rpm. Incubate for 2-3 hours until the absorbance at 600 nm reaches 0.5, cool the temperature of the culture to 30 ° C, add 0.2 mM IPTG, incubate overnight at 30 ° C, induce the expression of each protein, E. coli was recovered.
（２−２）大腸菌からのライセートの回収
前記（２−１）で得た大腸菌からライセートを抽出するために、1mM ABSF、protease inhibitor入りのPBSを2mL加え、超音波で大腸菌を破砕し、20000r.p.mで30分間遠心し、上清を回収し大腸菌ライセートとした。
(2-2) Recovery of lysate from E. coli In order to extract lysate from E. coli obtained in (2-1) above, 2 mL of PBS containing 1 mM ABSF and protease inhibitor was added, and E. coli was disrupted by ultrasonication. Centrifugation was performed at .pm for 30 minutes, and the supernatant was recovered to obtain Escherichia coli lysate.
（２−３）大腸菌からのペリプラズム画分の回収
前記（２−１）で得た大腸菌からペリプラズム画分を抽出するために、PE Buffer（20%Sucrose, 1mM EDTA, 100mM Tris-HCl pH8.0）を2mL加え、氷中で30分処理し、4000r.p.mで20分間遠心後、上清を回収しペリプラズム画分とした。
(2-3) Recovery of periplasmic fraction from E. coli In order to extract the periplasmic fraction from E. coli obtained in (2-1) above, PE Buffer (20% Sucrose, 1 mM EDTA, 100 mM Tris-HCl pH8.0 2 mL), treated in ice for 30 minutes, centrifuged at 4000 rpm for 20 minutes, and the supernatant was recovered to obtain a periplasm fraction.
（２−４）大腸菌からのサイトプラズム画分の回収
サイトプラズム画分を抽出するために、前記（２−３）で得たペリプラズム画分を除いた大腸菌に、1mM ABSF、protease inhibitor入りのPBSを2mL加え、超音波で大腸菌を完全に破砕し、20000r.p.mで30分間遠心後、上清を回収しサイトプラズム画分とした。
(2-4) Recovery of cytoplasmic fraction from E. coli In order to extract the cytoplasmic fraction, E. coli excluding the periplasmic fraction obtained in (2-3) above contains 1 mM ABSF and protease inhibitor. 2 mL of PBS was added, and E. coli was completely crushed with ultrasonic waves. After centrifugation at 20000 rpm for 30 minutes, the supernatant was collected and used as the cytoplasm fraction.
（２−５）ELISAによるA10B scFv発現大腸菌ライセートからのA10B scFvの結合活性測定
発現させたA10B scFv、A10B scFv-Zif268、A10B scFv-MBPの結合活性はELISAにより測定した。抗原（Rabbit IgG）をコーティングバッファーで10ng/mlに調整し、96wellイムノプレートに50μL/well分注し、室温で2時間コーティングした。コーティング後、プレートをPBSで2回洗浄し、2％BSAを250μL/well分注し室温で2時間ブロッキングした。
次いで、前記（２−３）及び（２−４）でそれぞれ分画したペリプラズム及びサイトプラズムを50μL/well分注し、室温で2時間抗原と反応させた。その後、PBS-T（0.05％ Tween20/PBS）で3回洗浄後、PBSで2回洗浄し、0.2％BSA/PBSで5000倍に希釈したHRPが結合した抗His抗体（Roche社）を1時間反応させ、PBS-T（0.05％ Tween20/PBS）で3回洗浄後、PBSで2回洗浄し、発色基質（Sigma社）と反応させた後、発色を測定しA10B scFvの結合活性を測定した。結果を図２に示す。
結果、発現させた全てのA10B scFvが結合活性を有することが確認された。A10B scFv単独及びA10B scFvとMPBを融合させたタンパク質は同程度の結合活性を有しており、Zif268を融合させた場合はA10B scFv単独及びA10B scFvとMPBを融合させたタンパク質と比較しておよそ100倍高い結合活性が確認された。
(2-5) A10B scFv expression by ELISA Measurement of binding activity of A10B scFv from E. coli lysate The binding activities of the expressed A10B scFv, A10B scFv-Zif268, and A10B scFv-MBP were measured by ELISA. Antigen (Rabbit IgG) was adjusted to 10 ng / ml with a coating buffer, dispensed into a 96-well immunoplate at 50 μL / well, and coated at room temperature for 2 hours. After coating, the plate was washed twice with PBS, and 2% BSA was dispensed at 250 μL / well and blocked at room temperature for 2 hours.
Next, 50 μL / well of periplasm and cytoplasm fractionated in the above (2-3) and (2-4) were dispensed and reacted with the antigen at room temperature for 2 hours. Then, wash 3 times with PBS-T (0.05% Tween20 / PBS), then 2 times with PBS, and anti-His antibody (Roche) bound to HRP diluted 5000 times with 0.2% BSA / PBS for 1 hour. After reacting, washed 3 times with PBS-T (0.05% Tween20 / PBS), washed 2 times with PBS and reacted with a chromogenic substrate (Sigma), color development was measured and the binding activity of A10B scFv was measured. . The results are shown in FIG.
As a result, it was confirmed that all expressed A10B scFv had binding activity. A10B scFv alone and A10B scFv and MPB-fused proteins have similar binding activity.When Zif268 is fused, the A10B scFv alone and A10B scFv alone and A10B scFv and MPB-fused proteins are roughly compared. A 100-fold higher binding activity was confirmed.
（実施例３）A10Bのペリプラズムとサイトプラズムでの発現及び結合活性の測定
（３−１）ペリプラズムの分画
実施例１で示した発現ベクター、pET-22b/A10B scFv、pET-22b/A10B scFv-Zif268、pET-30a/A10B scFv-Zif268、pET-22b/A10B scFv-MBP、を実施例２（２−１）と同様にタンパク質の発現を誘導した。発現誘導した大腸菌のペリプラズムからタンパク質を抽出するために、実施例２（２−３）と同様に、PE Buffer（20% Sucrose、100mMTris-HCl pH8.0、1mM EDTA）を2mL加え、15分間氷冷後、13000r.p.mで20分間遠心し、上清を回収しペリプラズムを分画した。
(Example 3) Expression of periplasm and cytoplasm of A10B and measurement of binding activity (3-1) Fraction of periplasm The expression vectors, pET-22b / A10B scFv, pET-22b / A10B shown in Example 1 In the same manner as in Example 2 (2-1), protein expression was induced for scFv-Zif268, pET-30a / A10B scFv-Zif268, and pET-22b / A10B scFv-MBP. In order to extract the protein from the periplasm of E. coli induced expression, 2 mL of PE Buffer (20% Sucrose, 100 mM Tris-HCl pH 8.0, 1 mM EDTA) was added as in Example 2 (2-3), and iced for 15 minutes. After cooling, the mixture was centrifuged at 13000 rpm for 20 minutes, the supernatant was collected, and the periplasm was fractionated.
（３−２）サイトプラズムの分画
前記（３−１）でペリプラズムを回収した大腸菌のペレットに、実施例２（２−４）と同様に、1mM ABSF、Protease inhibitorを加えたPBSを2mL加え、超音波で大腸菌を破砕し、20000r.p.mで30分間遠心し、上清を回収しサイトプラズムを分画した。
(3-2) Fractionation of cytoplasm In the same manner as in Example 2 (2-4), 2 mL of PBS containing 1 mM ABSF and protease inhibitor was added to the E. coli pellet from which the periplasm was recovered in (3-1). In addition, E. coli was disrupted with ultrasonic waves and centrifuged at 20000 rpm for 30 minutes. The supernatant was recovered and the cytoplasm was fractionated.
（３−３）ウェスタンブロッティングによる発現の確認
各A10B scFvのペリプラズム及びサイトプラズムの発現量はSDS-PAGE後にウェスタンブッティングによって発現量の比較を行なった。ペリプラズム及びサイトプラズム画分は各20μLとサンプルバッファー20μLを混合し、95℃、5分間熱処理し、10％アクリルアミドゲルで電気泳動した。電気泳動後のアクリルアミドゲルからタンパク質をトランスブロッターを用いてPDFメンブレン（ミリポア社）に転写し、5％スキムミルク/PBSでブロッキングした。ブロッキングしたメンブレンを0.5％のスキムミルクで5000倍に希釈したHRPが結合した抗His抗体（Roche社）によって1時間処理し、PBS-T（0.05％ Tween20/PBS）で3回洗浄後、PBSで2回洗浄し、発光基質（ミリポア社）と反応させた後、発光を測定することによりタンパク質の発現量を測定した。結果を図３Ａに示す。
pET-22b/A10B scFv、pET-22b/A10B scFv-MBP発現ベクターによって発現したタンパク質はpelBリーダー配列によりペリプラズムにタンパク質が移行しており、サイトプラズム内での移行途中のタンパク質も確認された。また、両タンパク質は同程度の発現量であることが分かった。
一方、pET-22b/A10B scFv-Zif268、pET-30a/A10B scFv-Zif268発現ベクターによって発現したタンパク質の場合は、pelBリーダー配列の無いpET-30a/A10B scFv-Zif268のみならず、pelBリーダー配列があるpET-22b/A10B scFv-Zif268においても、ペリプラズムには移行しておらず、発現したタンパク質がサイトプラズムに留まることが分かった。そして、Zif268を付加した場合は、いずれの場合でも発現量が飛躍的に向上することが示された。MBPと融合させた場合は、そのようなサイトプラズムに留まる現象も、発現増強効果も観察できなかった。
なお、N端にZif268を繋いだ場合（pET-22b/Zif268-A10B scFv発現ベクター）についても、同様の実験を行ったが、融合タンパク質の発現は全く観察されなかった。（結果は図示せず。）
(3-3) Confirmation of expression by Western blotting The expression level of each A10B scFv periplasm and cytoplasm was compared by Western butting after SDS-PAGE. For the periplasm and cytoplasm fractions, 20 μL each and 20 μL sample buffer were mixed, heat-treated at 95 ° C. for 5 minutes, and electrophoresed on a 10% acrylamide gel. Proteins were transferred from the acrylamide gel after electrophoresis to a PDF membrane (Millipore) using a transblotter and blocked with 5% skim milk / PBS. The blocked membrane was treated with anti-His antibody (Roche) conjugated with HRP diluted 5000 times with 0.5% skim milk for 1 hour, washed 3 times with PBS-T (0.05% Tween20 / PBS), and then 2 times with PBS. After washing twice and reacting with a luminescent substrate (Millipore), the expression level of the protein was measured by measuring luminescence. The results are shown in FIG. 3A.
The proteins expressed by the pET-22b / A10B scFv and pET-22b / A10B scFv-MBP expression vectors were transferred to the periplasm by the pelB leader sequence, and the proteins being transferred in the cytoplasm were also confirmed. In addition, both proteins were found to have similar expression levels.
On the other hand, in the case of proteins expressed by pET-22b / A10B scFv-Zif268 and pET-30a / A10B scFv-Zif268 expression vectors, not only pET-30a / A10B scFv-Zif268 without pelB leader sequence, but also pelB leader sequence Also in a certain pET-22b / A10B scFv-Zif268, it was found that the expressed protein remained in the cytoplasm without shifting to the periplasm. When Zif268 was added, the expression level was shown to be dramatically improved in any case. When it was fused with MBP, neither the phenomenon that remained in such cytoplasm nor the expression enhancing effect could be observed.
The same experiment was performed with Zif268 linked to the N-terminus (pET-22b / Zif268-A10B scFv expression vector), but no expression of the fusion protein was observed. (The result is not shown.)
（３−４）ELISAによる活性測定
各A10B scFvのペリプラズム及びサイトプラズムの結合活性は実施例２（２−５）と同様にELISAによって測定した。結果を図３Ｂに示す。
A10B scFvの結合活性も前記（３−３）の発現量と対応した結果が得られ、pET-22b/A10B、pET-22b/A10B-MBP発現ベクターによって発現したタンパク質は、どちらもペリプラズム及びサイトプラズム共に同等の結合活性を示した。pET-22b/A10B scFv-Zif268、pET-30a/A10B scFv-Zif268発現ベクターによって発現したタンパク質は、ペリプラズムにおける結合活性は全く見られず、サイトプラズムでは高い結合活性が示された。
(3-4) Activity measurement by ELISA The periplasmic and cytoplasmic binding activities of each A10B scFv were measured by ELISA in the same manner as in Example 2 (2-5). The results are shown in FIG. 3B.
The binding activity of A10B scFv also obtained a result corresponding to the expression level of (3-3), and the proteins expressed by the pET-22b / A10B and pET-22b / A10B-MBP expression vectors are both periplasm and cytoplasm. Showed the same binding activity. The proteins expressed by the pET-22b / A10B scFv-Zif268 and pET-30a / A10B scFv-Zif268 expression vectors showed no binding activity in the periplasm and high binding activity in the cytoplasm.
（実施例４）培養条件の変化によるA10B scFv及びA10B scFv-Zif268の結合活性測定
実施例２（２−１）で得たpET-22b/A10B scFv、pET-22b/A10B scFv-Zif268をBL21（DE3）へ形質転換したクローンをアンピシリンを添加したLB培地2mL、37℃、200r.p.mで一晩前培養する。前培養液1mLをアンピシリンを添加したLB培地100mLに加え、37℃、200r.p.mで培養する。波長600nmの吸光度が0.5に達するまで2〜3時間培養し、培養液の温度を30℃及び25℃に冷し、各々の温度の培養液に200μM及び50μMとなるようIPTGを加え、一晩培養し、各タンパク質の発現を誘導し、大腸菌を回収し、ライセートを回収後、実施例２（２−５）と同様にELISAによるA10B scFv発現大腸菌ライセートからのA10B scFvの結合活性を測定した。結果を図４に示す。
A10B scFvの発現は30℃、IPTG 200μMの条件で最も高い活性を示したが、その他の条件でも顕著な活性の変化は見られなかった。A10B scFv-Zif268の発現もA10B scFvの発現と同様の傾向を示し、培養条件によって大きな変化は見られないが、30℃、IPTG 200μMの条件で最も高い活性を示した。
(Example 4) Measurement of binding activity of A10B scFv and A10B scFv-Zif268 by changing culture conditions pET-22b / A10B scFv and pET-22b / A10B scFv-Zif268 obtained in Example 2 (2-1) were treated with BL21 ( The clone transformed into DE3) is pre-cultured overnight in 2 mL of LB medium supplemented with ampicillin at 37 ° C. and 200 rpm. 1 mL of the preculture is added to 100 mL of LB medium supplemented with ampicillin and cultured at 37 ° C. and 200 rpm. Incubate for 2-3 hours until the absorbance at 600 nm reaches 0.5, cool the temperature of the culture to 30 ° C and 25 ° C, add IPTG to the culture at each temperature to 200 µM and 50 µM, and culture overnight. Then, after the expression of each protein was induced, E. coli was recovered, and the lysate was recovered, the binding activity of A10B scFv from the A10B scFv expression E. coli lysate by ELISA was measured in the same manner as in Example 2 (2-5). The results are shown in FIG.
The expression of A10B scFv showed the highest activity under the conditions of 30 ° C. and IPTG 200 μM, but no significant change in activity was observed under other conditions. The expression of A10B scFv-Zif268 also showed the same tendency as the expression of A10B scFv, and no significant change was observed depending on the culture conditions, but the highest activity was observed under the conditions of 30 ° C. and IPTG 200 μM.
（実施例５）A10B scFv及びA10B scFv-Zif268の定量的な結合活性測定
（５−１）A10B scFv及びA10B scFv-Zif268の精製
A10B scFvを発現させた大腸菌のペリプラズムを分画し、A10B scFv-Zif268を発現させた大腸菌のサイトプラズムを分画した。各々の画分からニッケルカラム（HisTrap FF GE社）を用いてGE社のプロトコールに従い、A10B scFv、A10B scFv-Zif268を精製した。精製したタンパク質はProtein assay kit（ピアス社）でタンパク質濃度を測定し、定量した。それぞれの収量はA10B scFvが0.15mg/L、A10B scFv-Zif268が12.3mg/Lであった。
(Example 5) Quantitative binding activity measurement of A10B scFv and A10B scFv-Zif268 (5-1) Purification of A10B scFv and A10B scFv-Zif268
The periplasm of E. coli expressing A10B scFv was fractionated, and the cytoplasm of E. coli expressing A10B scFv-Zif268 was fractionated. A10B scFv and A10B scFv-Zif268 were purified from each fraction using a nickel column (HisTrap FF GE) according to the protocol of GE. The purified protein was quantified by measuring the protein concentration with a Protein assay kit (Pierce). Each yield was 0.15 mg / L for A10B scFv and 12.3 mg / L for A10B scFv-Zif268.
（５−２）A10B scFv及びA10B scFv-Zif268の結合活性測定
前記（５−１）で精製したA10B scFv及びA10B scFv-Zif268を実施例２（２−５）と同様にELISAにより定量的な結合の活性を測定した。結果を図５に示す。
精製したA10B scFv及びA10B scFv-Zif268の分子量あたりの活性は同程度であることが示された。この結果から、Zif268をA10Bに融合することによりその発現量をサイトプラズムにおいて向上させる。また、通常scFvはペリプラズムに移行することで立体構造を形成し結合活性を発揮することが知られているが、Zif268を融合することによってサイトプラズム内で結合活性を持つことが明らかとなった。
(5-2) Measurement of binding activity of A10B scFv and A10B scFv-Zif268 The amount of A10B scFv and A10B scFv-Zif268 purified in (5-1) was quantitatively bound by ELISA in the same manner as in Example 2 (2-5). The activity of was measured. The results are shown in FIG.
It was shown that the activity per molecular weight of the purified A10B scFv and A10B scFv-Zif268 was comparable. From this result, the expression level is improved in cytoplasm by fusing Zif268 to A10B. In addition, it is known that scFv normally forms a three-dimensional structure by moving to the periplasm and exhibits binding activity, but it has been revealed that it has binding activity in the cytoplasm by fusing Zif268. .
（実施例６）Zif268によるA10B scFv以外のscFvへの発現増強効果
実施例１（１−１）、（１−２）、（１−５）、（１−６）及び（１−７）で作製した下記の発現ベクターを用いて、実施例２と同様の方法により大腸菌で発現させ、実施例２（２−３）、（２−４）と同様の方法により分画したペリプラズム画分及びサイトプラズム画分を用い、実施例２と同様の方法により、ELISAによってZif268のA10B以外のscFvの発現に対する効果を測定した。
（１）pET-22b/A10B scFv及びpET-22b/A10B scFv-Zif268
（２）pET-22b/Mouse anti-GLuc scFv及びpET-22b/Mouse anti-GLuc scFv-Zif268
（３）pET-22b/Chicken anti-rabbit IgG scFv及びpET-22b/Chicken anti-rabbit IgG scFv-Zif268
その結果、A10B scFv以外のscFvの場合も、scFv単独よりもZif268を付加して発現させた場合には、サイトプラズマに留まることが確認され、かつscFv活性の向上が見られた。この結果から、Zif268はどのような種類のscFvに対しても、そのC末端側に融合させた融合タンパクとして細菌で発現させることで、その産生を増強し、サイトプラズム内においてscFv活性を有するscFvを発現させることが可能となることが示された。
(Example 6) Expression enhancement effect by Zif268 on scFv other than A10B scFv In Example 1 (1-1), (1-2), (1-5), (1-6) and (1-7) Periplasm fractions and sites that were expressed in E. coli by the same method as in Example 2 using the prepared expression vector below and fractionated in the same manner as in Examples 2 (2-3) and (2-4) Using the plasma fraction, the effect of Zif268 on the expression of scFv other than A10B was measured by ELISA in the same manner as in Example 2.
(1) pET-22b / A10B scFv and pET-22b / A10B scFv-Zif268
(2) pET-22b / Mouse anti-GLuc scFv and pET-22b / Mouse anti-GLuc scFv-Zif268
(3) pET-22b / Chicken anti-rabbit IgG scFv and pET-22b / Chicken anti-rabbit IgG scFv-Zif268
As a result, in the case of scFv other than A10B scFv, it was confirmed that when it was expressed by adding Zif268 rather than scFv alone, it remained in the cytoplasm, and the scFv activity was improved. From this result, Zif268 enhances the production of any kind of scFv in bacteria as a fusion protein fused to the C-terminal side, and has scFv activity in the cytoplasm It has been shown that it is possible to express scFv.
（実施例７）trpR及びHinRにおけるscFvへの発現増強効果
実施例１（１−１）、（１−２）、（１−８）及び（１−９）で作製した下記の発現ベクターを用いて、実施例２と同様の方法により大腸菌で発現させ、先に実施例２（２−２）と同様の方法によりライセートを回収し、ELISAによってZif268以外のDNA結合タンパク質のA10B発現に対する効果を測定した。結果を図７Ａに示す。次いで、実施例２（２−３）、（２−４）と同様の方法により分画したペリプラズム画分及びサイトプラズム画分を用い、実施例２と同様の方法により、ELISAによってZif268以外のDNA結合タンパク質のA10B発現に対する効果を測定した。結果を図７Ｂに示す。
（１）pET-22b/A10B scFv
（２）pET-22b/A10B scFv-Zif268
（３）pET-22b/A10B scFv-trpR
（４）pET-22b/A10B scFv-HinR
その結果、trpR又はHinRをA10Bに付加した場合も、Zif268の場合と同様に、A10B単独での発現と比較して発現の大きな向上が見られ、かつ細胞質内で発現していた。またtrpR又はHinRをA10Bに付加した場合は、発現しているscFv-trpRおよびscFv-HinRは、scFv-Zif268同様、高い抗原結合活性を示した。この結果から、Zif268と同様に、trpR及びHinRには、scFvのC末端側に融合させることで、細胞質内で、機能的なscFvの発現を増強させることが示された。
(Example 7) Expression enhancement effect on scFv in trpR and HinR Using the following expression vectors prepared in Example 1 (1-1), (1-2), (1-8) and (1-9) Then, it is expressed in E. coli by the same method as in Example 2, and the lysate is first recovered by the same method as in Example 2 (2-2), and the effect on the A10B expression of DNA binding proteins other than Zif268 is measured by ELISA. did. The results are shown in FIG. 7A. Subsequently, by using the periplasm fraction and the cytoplasm fraction fractionated by the same method as in Example 2 (2-3) and (2-4), the ELISA was performed by ELISA in the same manner as in Example 2 except for Zif268. The effect of DNA binding protein on A10B expression was measured. The result is shown in FIG. 7B.
(1) pET-22b / A10B scFv
(2) pET-22b / A10B scFv-Zif268
(3) pET-22b / A10B scFv-trpR
(4) pET-22b / A10B scFv-HinR
As a result, when trpR or HinR was added to A10B, similar to the case of Zif268, the expression was greatly improved compared to the expression of A10B alone, and it was expressed in the cytoplasm. In addition, when trpR or HinR was added to A10B, the expressed scFv-trpR and scFv-HinR showed high antigen binding activity, similar to scFv-Zif268. From this result, it was shown that, like Zif268, trpR and HinR were fused to the C-terminal side of scFv to enhance the expression of functional scFv in the cytoplasm.
（実施例８）Zif268によるscFv以外のタンパク質への発現増強効果
Zif268の各種scFv以外の外来タンパク質への発現増強効果を確認するため、実施例１（１−１０）、（１−１１）、（１−１２）及び（１−１３）で作製した抗Gaussiaルシフェラーゼ（GLuc)-Zif268及びCD8-Zif268の発現ベクターを用いて、実施例２と同様の方法により大腸菌で発現させ、実施例２（２−３）、（２−４）と同様の方法により分画したペリプラズム画分及びサイトプラズム画分を用い、実施例２と同様の方法により、ELISAによりGLuc及びGLuc-Zif268とCD8及びCD8-Zif268の活性を測定した。結果を図８に示す。
その結果、GLuc及びCD8両タンパク質共に、A10B scFvの場合と同様に、Zif268を付加することによって細胞質内で発現しており、かつGLuc又はCD8単独での発現と比較して活性の向上が見られた。この結果により、Zif268はscFvのみならず、どのような外来タンパク質であっても、そのC末端側に融合させることで、細胞質内で、機能的な活性を保持した外来タンパク質の産生を増強させることが示された。
(Example 8) Expression enhancement effect on proteins other than scFv by Zif268
Anti-Gaussia luciferase prepared in Example 1 (1-10), (1-11), (1-12) and (1-13) to confirm the effect of enhancing the expression of Zif268 on foreign proteins other than various scFvs Using the expression vectors of (GLuc) -Zif268 and CD8-Zif268, expression is performed in E. coli by the same method as in Example 2, and fractionation is performed by the same method as in Examples 2 (2-3) and (2-4). Using the periplasm fraction and cytoplasm fraction, the activities of GLuc and GLuc-Zif268 and CD8 and CD8-Zif268 were measured by ELISA in the same manner as in Example 2. The results are shown in FIG.
As a result, both GLuc and CD8 proteins were expressed in the cytoplasm by adding Zif268, as in the case of A10B scFv, and improved in activity compared to the expression of GLuc or CD8 alone. It was. As a result, Zif268 enhances the production of foreign proteins that retain functional activity in the cytoplasm by fusing not only scFv but any foreign protein to the C-terminal side. It has been shown.
１．配列番号１：Zif268
２．配列番号２：Zif268（gene）
３．配列番号３：HinR
４．配列番号４：HinR（gene）
５．配列番号５：Tryptophan transcriptional repressor（trpR）E.Coli
６．配列番号６：trpR（gene）
７．配列番号７：A10B（Mouse anti-rabbit IgG）scFv（gene）
８．配列番号８：forward primer（A10B scFv-Zif268）
９．配列番号９：reverse primer（A10B scFv-Zif268）
１０．配列番号１０：forward primer（A10B scFv-MBP）
１１．配列番号１１：reverse primer（A10B scFv-MBP）
１２．配列番号１２：Anti GLuc scFv（GL-11-6）
１３．配列番号１３：forward primer（Anti GLuc scFv-Zif268）
１４．配列番号１４：reverse primer（Anti GLuc scFv-Zif268）
１５．配列番号１５：Chicken anti-rabbit IgG scFv
１６．配列番号１６：Gaussia luciferase（GLuc）
１７．配列番号１７：Human CD8α
１８．配列番号１８：forward primer（CD8）
１９．配列番号１９：reverse primer（CD8）
1. Sequence number 1: Zif268
2. Sequence number 2: Zif268 (gene)
3. Sequence number 3: HinR
4). SEQ ID NO: 4: HinR (gene)
5. Sequence number 5: Tryptophan transcriptional repressor (trpR) E.Coli
6). SEQ ID NO: 6: trpR (gene)
7). SEQ ID NO: 7: A10B (Mouse anti-rabbit IgG) scFv (gene)
8). Sequence number 8: forward primer (A10B scFv-Zif268)
9. Sequence number 9: reverse primer (A10B scFv-Zif268)
10. SEQ ID NO: 10: forward primer (A10B scFv-MBP)
11. Sequence number 11: reverse primer (A10B scFv-MBP)
12 Sequence number 12: Anti GLuc scFv (GL-11-6)
13. SEQ ID NO: 13: forward primer (Anti GLuc scFv-Zif268)
14 Sequence number 14: reverse primer (Anti GLuc scFv-Zif268)
15. SEQ ID NO: 15: Chicken anti-rabbit IgG scFv
16. Sequence number 16: Gaussia luciferase (GLuc)
17. SEQ ID NO: 17: Human CD8α
18. SEQ ID NO: 18: forward primer (CD8)
19. Sequence number 19: reverse primer (CD8)
Claims (8)
ここで、Zif268 DNA結合ドメインは、配列番号１、もしくはその１ないし５個のアミノ酸が欠失・置換・挿入・付加されたアミノ酸配列で示されるタンパク質であり、trpR DNA結合ドメインは、配列番号５、もしくはその１ないし１０個のアミノ酸が欠失・置換・挿入・付加されたアミノ酸配列で示されるタンパク質であり、またHinR DNA結合ドメインは、配列番号３、もしくはその１ないし５個のアミノ酸が欠失・置換・挿入・付加されたアミノ酸配列に示されるタンパク質である、方法。 The C-terminal of the desired foreign protein, characterized in that the bacterial host transformed with an expression vector comprising a Zif268, trpR, and nucleic acid encoding a fusion protein fused with selected DNA binding domain from HinR A method for enhancing the expression of a functional foreign protein in the bacterial host cytoplasm ,
Here, the Zif268 DNA binding domain is SEQ ID NO: 1 or a protein represented by an amino acid sequence in which 1 to 5 amino acids are deleted, substituted, inserted, or added, and the trpR DNA binding domain is SEQ ID NO: 5 Or a protein represented by an amino acid sequence in which 1 to 10 amino acids are deleted, substituted, inserted, or added, and the HinR DNA binding domain lacks SEQ ID NO: 3 or 1 to 5 amino acids thereof. A method, which is a protein represented by a deleted, substituted, inserted, or added amino acid sequence .
ここで、Zif268 DNA結合ドメインは、配列番号１、もしくはその１ないし５個のアミノ酸が欠失・置換・挿入・付加されたアミノ酸配列で示されるタンパク質であり、trpR DNA結合ドメインは、配列番号５、もしくはその１ないし１０個のアミノ酸が欠失・置換・挿入・付加されたアミノ酸配列で示されるタンパク質であり、またHinR DNA結合ドメインは、配列番号３、もしくはその１ないし５個のアミノ酸が欠失・置換・挿入・付加されたアミノ酸配列に示されるタンパク質である、機能的外来タンパク質発現増強剤。 The C-terminal of the desired foreign protein, Zif268, trpR, and as an active ingredient a nucleic acid encoding a fusion protein fused to selected DNA binding domain from HinR, functional foreign protein in a bacterial host cell cytoplasm An expression enhancer comprising :
Here, the Zif268 DNA binding domain is SEQ ID NO: 1 or a protein represented by an amino acid sequence in which 1 to 5 amino acids are deleted, substituted, inserted, or added, and the trpR DNA binding domain is SEQ ID NO: 5 Or a protein represented by an amino acid sequence in which 1 to 10 amino acids are deleted, substituted, inserted, or added, and the HinR DNA binding domain lacks SEQ ID NO: 3 or 1 to 5 amino acids thereof. A functional foreign protein expression enhancer, which is a protein represented by a deleted, substituted, inserted, or added amino acid sequence .
ここで、Zif268 DNA結合ドメインは、配列番号１、もしくはその１ないし５個のアミノ酸が欠失・置換・挿入・付加されたアミノ酸配列で示されるタンパク質であり、trpR DNA結合ドメインは、配列番号５、もしくはその１ないし１０個のアミノ酸が欠失・置換・挿入・付加されたアミノ酸配列で示されるタンパク質であり、またHinR DNA結合ドメインは、配列番号３、もしくはその１ないし５個のアミノ酸が欠失・置換・挿入・付加されたアミノ酸配列に示されるタンパク質である、使用。 Zif268, trpR, and to the use of nucleic acid encoding the selected DNA binding domain from HinR, by a nucleic acid encoding the DNA-binding protein, it is fused to the 3 'end of the nucleic acid encoding the antibody fragment, the antibody fragment to the use for expression as functional antibody fragments in the cytoplasm of the bacterial host,
Here, the Zif268 DNA binding domain is SEQ ID NO: 1 or a protein represented by an amino acid sequence in which 1 to 5 amino acids are deleted, substituted, inserted, or added, and the trpR DNA binding domain is SEQ ID NO: 5 Or a protein represented by an amino acid sequence in which 1 to 10 amino acids are deleted, substituted, inserted, or added, and the HinR DNA binding domain lacks SEQ ID NO: 3 or 1 to 5 amino acids thereof. Use, which is a protein represented by a deleted, substituted, inserted, or added amino acid sequence .
（ａ）Zif268、trpR、及びHinRから選択されたDNA結合ドメインをコードする第１の核酸分子を、前記外来タンパク質をコードする第２の核酸分子の3’末端側に融合させて、細菌宿主における発現産物が、前記DNA結合タンパク質が前記外来タンパク質のＣ末側に位置するような融合タンパク質をコードする遺伝子構築物を形成する工程であって、
ここで、Zif268 DNA結合ドメインは、配列番号１、もしくはその１ないし５個のアミノ酸が欠失・置換・挿入・付加されたアミノ酸配列で示されるタンパク質であり、trpR DNA結合ドメインは、配列番号５、もしくはその１ないし１０個のアミノ酸が欠失・置換・挿入・付加されたアミノ酸配列で示されるタンパク質であり、またHinR DNA結合ドメインは、配列番号３、もしくはその１ないし５個のアミノ酸が欠失・置換・挿入・付加されたアミノ酸配列に示されるタンパク質である、工程、
（ｂ）前記構築物を、細菌宿主の細胞質内で発現させる工程。 A method for enhancing the expression level of a desired foreign protein in a bacterial host, comprising the following steps (a) and (b):
(A) Zif268, trpR, and the first nucleic acid molecule encoding a selected DNA binding domain from HinR, and said fused to the 3 'end of the second nucleic acid molecule encoding a foreign protein, the bacterial host Wherein the expression product in (i) forms a gene construct encoding a fusion protein such that the DNA binding protein is located on the C-terminal side of the foreign protein ,
Here, the Zif268 DNA binding domain is SEQ ID NO: 1 or a protein represented by an amino acid sequence in which 1 to 5 amino acids are deleted, substituted, inserted, or added, and the trpR DNA binding domain is SEQ ID NO: 5 Or a protein represented by an amino acid sequence in which 1 to 10 amino acids are deleted, substituted, inserted, or added, and the HinR DNA binding domain lacks SEQ ID NO: 3 or 1 to 5 amino acids thereof. A process represented by a deleted, substituted, inserted, or added amino acid sequence ,
(B) expressing the construct in the cytoplasm of a bacterial host.
（ｃ）発現させた融合タンパク質を宿主細菌の細胞質画分から回収する工程。 Furthermore, the method of Claim 6 including the following process (c);
(C) recovering the expressed fusion protein from the cytoplasmic fraction of the host bacterium.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2013240294A JP6303195B2 (en) | 2013-11-20 | 2013-11-20 | Method for producing functional foreign protein by bacteria |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2013240294A JP6303195B2 (en) | 2013-11-20 | 2013-11-20 | Method for producing functional foreign protein by bacteria |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JP2015097519A JP2015097519A (en) | 2015-05-28 |
| JP6303195B2 true JP6303195B2 (en) | 2018-04-04 |
Family
ID=53374605
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2013240294A Active JP6303195B2 (en) | 2013-11-20 | 2013-11-20 | Method for producing functional foreign protein by bacteria |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP6303195B2 (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| TW201814045A (en) * | 2016-09-16 | 2018-04-16 | 英商艾普森生物製藥有限公司 | Method for producing di-chain clostridial neurotoxins |
| CN110396533B (en) * | 2019-07-04 | 2023-05-26 | 成都英普博集生物科技有限公司 | Escherichia coli strain with phage resistance, screening method and preparation method of recombinant engineering bacterium expressed recombinant protein |
| CA3227440A1 (en) | 2021-07-27 | 2023-02-02 | Stand Therapeutics Co., Ltd. | Peptide tag and nucleic acid encoding same |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| MX2007013757A (en) * | 2005-05-05 | 2008-01-24 | Univ Arizona | Sequence enabled reassembly (seer) - a novel method for visualizing specific dna sequences. |
| US20080311047A1 (en) * | 2005-11-16 | 2008-12-18 | Thijs Kaper | Multimetric Biosensors and Methods of Using Same |
| KR20090031938A (en) * | 2006-07-05 | 2009-03-30 | 더 스크립스 리서치 인스티튜트 | Chimeric zinc finger recombinases optimized for catalysis by directed evolution |
-
2013
- 2013-11-20 JP JP2013240294A patent/JP6303195B2/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| JP2015097519A (en) | 2015-05-28 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP6181145B2 (en) | Novel immunoglobulin binding proteins with improved specificity | |
| Sun et al. | A combined strategy improves the solubility of aggregation-prone single-chain variable fragment antibodies | |
| Klatt et al. | Secretory signal peptide modification for optimized antibody-fragment expression-secretion in Leishmania tarentolae | |
| Wang et al. | Engineering production of functional scFv antibody in E. coli by co-expressing the molecule chaperone Skp | |
| Zhou et al. | Enhancing full-length antibody production by signal peptide engineering | |
| EP2420253A1 (en) | Engineering multifunctional and multivalent molecules with collagen XV trimerization domain | |
| CA2640671A1 (en) | Engineered antibody-stress protein fusions | |
| JP2013509867A5 (en) | ||
| Sonoda et al. | Functional expression of single-chain Fv antibody in the cytoplasm of Escherichia coli by thioredoxin fusion and co-expression of molecular chaperones | |
| Hu et al. | Cloning, expression and characterisation of a single-chain Fv antibody fragment against domoic acid in Escherichia coli | |
| Akbari et al. | Functional expression of a single-chain antibody fragment against human epidermal growth factor receptor 2 (HER2) in Escherichia coli | |
| JP6303195B2 (en) | Method for producing functional foreign protein by bacteria | |
| WO2020188346A1 (en) | Antigen binding proteins | |
| JP2004510410A (en) | Leader peptide that promotes secretion of host cell-derived recombinant protein | |
| RU2742006C2 (en) | Method for producing recombinant protein | |
| Yousefi et al. | Soluble expression of humanized anti-CD20 single chain antibody in Escherichia coli by cytoplasmic chaperones co-expression | |
| Ye et al. | High-level expression and characterization of an anti-VEGF165 single-chain variable fragment (scFv) by small ubiquitin-related modifier fusion in Escherichia coli | |
| US20080280346A1 (en) | System For the Production of Dimeric Proteins Based on the Transport System of Hemolysin of Escherichia Coli | |
| Caswell et al. | Production of recombinant proteins in Escherichia coli using an N-terminal tag derived from sortase | |
| Mokhonov et al. | SlyD-deficient Escherichia coli strains: A highway to contaminant-free protein extraction | |
| Hanyu | Fusion of Zif268 to the C-Terminus of Scfvs Promotes Expression of the Active Form in the Cytoplasm of Escherichia coli | |
| Tapryal et al. | Cloning, expression and efficient refolding of carbohydrate–peptide mimicry recognizing single chain antibody 2D10 | |
| Zhu et al. | SHTXTHHly, an extracellular secretion platform for the preparation of bioactive peptides and proteins in Escherichia coli | |
| Trabzonlu et al. | Effects of different cytoplasmic chaperones on outer membrane solubility in Escherichia coli; rOMP25 modeling. | |
| JP2015187083A (en) | Protein-magnetic particle conjugate and production method thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20160908 |
|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20160915 |
|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20160908 |
|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20161019 |
|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20161205 |
|
| A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20171101 |
|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20171225 |
|
| 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: 20180126 |
|
| A61 | First payment of annual fees (during grant procedure) |
Free format text: JAPANESE INTERMEDIATE CODE: A61 Effective date: 20180213 |
|
| R150 | Certificate of patent or registration of utility model |
Ref document number: 6303195 Country of ref document: JP Free format text: JAPANESE INTERMEDIATE CODE: R150 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |