WO2019151901A1 - Method of producing recombinant beta-n-acetylglucosaminidase strh from streptococcus pneumoniae - Google Patents
Method of producing recombinant beta-n-acetylglucosaminidase strh from streptococcus pneumoniae Download PDFInfo
- Publication number
- WO2019151901A1 WO2019151901A1 PCT/RU2019/000045 RU2019000045W WO2019151901A1 WO 2019151901 A1 WO2019151901 A1 WO 2019151901A1 RU 2019000045 W RU2019000045 W RU 2019000045W WO 2019151901 A1 WO2019151901 A1 WO 2019151901A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- strh
- glcnacase
- chromatography
- protein
- acetylglucosaminidase
- Prior art date
Links
- 238000000034 method Methods 0.000 title claims abstract description 27
- 241000193998 Streptococcus pneumoniae Species 0.000 title description 12
- 229940031000 streptococcus pneumoniae Drugs 0.000 title description 10
- 108010055851 Acetylglucosaminidase Proteins 0.000 title 1
- 102100030122 Protein O-GlcNAcase Human genes 0.000 title 1
- 108090000623 proteins and genes Proteins 0.000 claims description 60
- 102000004169 proteins and genes Human genes 0.000 claims description 52
- 238000004587 chromatography analysis Methods 0.000 claims description 26
- 239000000872 buffer Substances 0.000 claims description 22
- 229920002684 Sepharose Polymers 0.000 claims description 21
- 241000588724 Escherichia coli Species 0.000 claims description 18
- 238000002523 gelfiltration Methods 0.000 claims description 15
- 238000005571 anion exchange chromatography Methods 0.000 claims description 11
- 102100021935 C-C motif chemokine 26 Human genes 0.000 claims description 10
- 101000897493 Homo sapiens C-C motif chemokine 26 Proteins 0.000 claims description 10
- 125000000484 butyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 claims description 9
- 239000013522 chelant Substances 0.000 claims description 8
- 108010093488 His-His-His-His-His-His Proteins 0.000 claims description 7
- 239000012564 Q sepharose fast flow resin Substances 0.000 claims description 7
- 229910052751 metal Inorganic materials 0.000 claims description 7
- 239000002184 metal Substances 0.000 claims description 7
- 239000002594 sorbent Substances 0.000 claims description 7
- 239000012505 Superdex™ Substances 0.000 claims description 6
- 239000012528 membrane Substances 0.000 claims description 5
- 238000000108 ultra-filtration Methods 0.000 claims description 5
- 230000002209 hydrophobic effect Effects 0.000 claims description 4
- 230000002378 acidificating effect Effects 0.000 claims description 2
- 238000011097 chromatography purification Methods 0.000 claims description 2
- 238000005406 washing Methods 0.000 claims description 2
- 125000003275 alpha amino acid group Chemical group 0.000 claims 1
- 238000002360 preparation method Methods 0.000 abstract description 47
- 238000004519 manufacturing process Methods 0.000 abstract description 18
- 238000004458 analytical method Methods 0.000 abstract description 11
- 102000003886 Glycoproteins Human genes 0.000 abstract description 8
- 108090000288 Glycoproteins Proteins 0.000 abstract description 8
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 abstract description 5
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 abstract description 5
- 244000005700 microbiome Species 0.000 abstract description 4
- 241001465754 Metazoa Species 0.000 abstract description 3
- 150000004676 glycans Chemical group 0.000 abstract description 2
- 238000007634 remodeling Methods 0.000 abstract description 2
- 230000001225 therapeutic effect Effects 0.000 abstract description 2
- 235000018102 proteins Nutrition 0.000 description 32
- 239000002158 endotoxin Substances 0.000 description 21
- 238000000746 purification Methods 0.000 description 19
- 238000012360 testing method Methods 0.000 description 19
- 108020004414 DNA Proteins 0.000 description 17
- 239000000758 substrate Substances 0.000 description 17
- 229940079593 drug Drugs 0.000 description 16
- 239000003814 drug Substances 0.000 description 16
- 239000000243 solution Substances 0.000 description 15
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 12
- 239000000523 sample Substances 0.000 description 12
- 102000005744 Glycoside Hydrolases Human genes 0.000 description 11
- 108010031186 Glycoside Hydrolases Proteins 0.000 description 11
- 230000000694 effects Effects 0.000 description 11
- 239000002028 Biomass Substances 0.000 description 10
- 238000004128 high performance liquid chromatography Methods 0.000 description 10
- 239000000047 product Substances 0.000 description 9
- 102000004190 Enzymes Human genes 0.000 description 8
- 108090000790 Enzymes Proteins 0.000 description 8
- 230000001580 bacterial effect Effects 0.000 description 8
- 230000015572 biosynthetic process Effects 0.000 description 8
- 229940088598 enzyme Drugs 0.000 description 8
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 description 8
- 230000003287 optical effect Effects 0.000 description 8
- 239000013612 plasmid Substances 0.000 description 8
- 125000000539 amino acid group Chemical group 0.000 description 7
- 238000010790 dilution Methods 0.000 description 7
- 239000012895 dilution Substances 0.000 description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 7
- 239000012480 LAL reagent Substances 0.000 description 6
- 210000004027 cell Anatomy 0.000 description 6
- 230000001413 cellular effect Effects 0.000 description 6
- 108020001507 fusion proteins Proteins 0.000 description 6
- 102000037865 fusion proteins Human genes 0.000 description 6
- 239000012535 impurity Substances 0.000 description 6
- 239000011780 sodium chloride Substances 0.000 description 6
- 108010074860 Factor Xa Proteins 0.000 description 5
- 230000007062 hydrolysis Effects 0.000 description 5
- 238000006460 hydrolysis reaction Methods 0.000 description 5
- 108090000765 processed proteins & peptides Proteins 0.000 description 5
- 239000012086 standard solution Substances 0.000 description 5
- BTJIUGUIPKRLHP-UHFFFAOYSA-N 4-nitrophenol Chemical compound OC1=CC=C([N+]([O-])=O)C=C1 BTJIUGUIPKRLHP-UHFFFAOYSA-N 0.000 description 4
- 108010024636 Glutathione Proteins 0.000 description 4
- 108010076504 Protein Sorting Signals Proteins 0.000 description 4
- 108090000190 Thrombin Proteins 0.000 description 4
- 238000009825 accumulation Methods 0.000 description 4
- 238000005349 anion exchange Methods 0.000 description 4
- 238000007865 diluting Methods 0.000 description 4
- 238000001962 electrophoresis Methods 0.000 description 4
- 238000010828 elution Methods 0.000 description 4
- 229960003180 glutathione Drugs 0.000 description 4
- 238000002955 isolation Methods 0.000 description 4
- 239000006166 lysate Substances 0.000 description 4
- 239000008213 purified water Substances 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 108091008146 restriction endonucleases Proteins 0.000 description 4
- 239000012089 stop solution Substances 0.000 description 4
- 229960004072 thrombin Drugs 0.000 description 4
- 239000013598 vector Substances 0.000 description 4
- 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 3
- 108020004705 Codon Proteins 0.000 description 3
- 108091028043 Nucleic acid sequence Proteins 0.000 description 3
- 238000011529 RT qPCR Methods 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 101710137500 T7 RNA polymerase Proteins 0.000 description 3
- 208000007536 Thrombosis Diseases 0.000 description 3
- 150000001413 amino acids Chemical group 0.000 description 3
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- 239000000356 contaminant Substances 0.000 description 3
- 239000012531 culture fluid Substances 0.000 description 3
- 239000013024 dilution buffer Substances 0.000 description 3
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 229910052760 oxygen Inorganic materials 0.000 description 3
- 239000001301 oxygen Substances 0.000 description 3
- 230000035945 sensitivity Effects 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- 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
- 241000196324 Embryophyta Species 0.000 description 2
- 102000004157 Hydrolases Human genes 0.000 description 2
- 108090000604 Hydrolases Proteins 0.000 description 2
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 2
- 239000007987 MES buffer Substances 0.000 description 2
- 108700005078 Synthetic Genes Proteins 0.000 description 2
- 229960000723 ampicillin Drugs 0.000 description 2
- 238000003149 assay kit Methods 0.000 description 2
- 230000003197 catalytic effect Effects 0.000 description 2
- 239000013592 cell lysate Substances 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 238000010367 cloning Methods 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 230000002255 enzymatic effect Effects 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 238000011194 good manufacturing practice Methods 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 235000014304 histidine Nutrition 0.000 description 2
- 125000000487 histidyl group Chemical class [H]N([H])C(C(=O)O*)C([H])([H])C1=C([H])N([H])C([H])=N1 0.000 description 2
- 239000008101 lactose Substances 0.000 description 2
- 229920006008 lipopolysaccharide Polymers 0.000 description 2
- 238000013507 mapping Methods 0.000 description 2
- 235000015097 nutrients Nutrition 0.000 description 2
- 229920001542 oligosaccharide Polymers 0.000 description 2
- 150000002482 oligosaccharides Chemical class 0.000 description 2
- 238000005457 optimization Methods 0.000 description 2
- YBYRMVIVWMBXKQ-UHFFFAOYSA-N phenylmethanesulfonyl fluoride Chemical compound FS(=O)(=O)CC1=CC=CC=C1 YBYRMVIVWMBXKQ-UHFFFAOYSA-N 0.000 description 2
- 239000013641 positive control Substances 0.000 description 2
- 238000003753 real-time PCR Methods 0.000 description 2
- DKVBOUDTNWVDEP-NJCHZNEYSA-N teicoplanin aglycone Chemical class N([C@H](C(N[C@@H](C1=CC(O)=CC(O)=C1C=1C(O)=CC=C2C=1)C(O)=O)=O)[C@H](O)C1=CC=C(C(=C1)Cl)OC=1C=C3C=C(C=1O)OC1=CC=C(C=C1Cl)C[C@H](C(=O)N1)NC([C@H](N)C=4C=C(O5)C(O)=CC=4)=O)C(=O)[C@@H]2NC(=O)[C@@H]3NC(=O)[C@@H]1C1=CC5=CC(O)=C1 DKVBOUDTNWVDEP-NJCHZNEYSA-N 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 108091071337 20 family Proteins 0.000 description 1
- 101710129874 ATPase PAAT Proteins 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 244000045232 Canavalia ensiformis Species 0.000 description 1
- 235000010520 Canavalia ensiformis Nutrition 0.000 description 1
- 108050001186 Chaperonin Cpn60 Proteins 0.000 description 1
- 102000052603 Chaperonins Human genes 0.000 description 1
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 1
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 1
- 241001025319 Etheostoma collis Species 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 101710142847 Glutathione S-transferase class-mu 26 kDa isozyme Proteins 0.000 description 1
- 108010070675 Glutathione transferase Proteins 0.000 description 1
- 102000005720 Glutathione transferase Human genes 0.000 description 1
- 229930186217 Glycolipid Natural products 0.000 description 1
- 102000002812 Heat-Shock Proteins Human genes 0.000 description 1
- 108010004889 Heat-Shock Proteins Proteins 0.000 description 1
- 229920000663 Hydroxyethyl cellulose Polymers 0.000 description 1
- 102000003960 Ligases Human genes 0.000 description 1
- 108090000364 Ligases Proteins 0.000 description 1
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 description 1
- 125000003047 N-acetyl group Chemical group 0.000 description 1
- OVRNDRQMDRJTHS-RTRLPJTCSA-N N-acetyl-D-glucosamine Chemical group CC(=O)N[C@H]1C(O)O[C@H](CO)[C@@H](O)[C@@H]1O OVRNDRQMDRJTHS-RTRLPJTCSA-N 0.000 description 1
- 208000025174 PANDAS Diseases 0.000 description 1
- 208000021155 Paediatric autoimmune neuropsychiatric disorders associated with streptococcal infection Diseases 0.000 description 1
- 240000004718 Panda Species 0.000 description 1
- 235000016496 Panda oleosa Nutrition 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 108090000279 Peptidyltransferases Proteins 0.000 description 1
- 206010035664 Pneumonia Diseases 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 239000012506 Sephacryl® Substances 0.000 description 1
- 102100033869 Sodium-coupled neutral amino acid transporter 4 Human genes 0.000 description 1
- 241000194017 Streptococcus Species 0.000 description 1
- 101150114197 TOP gene Proteins 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 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 1
- 238000012443 analytical study Methods 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 239000012490 blank solution Substances 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 150000001720 carbohydrates Chemical group 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 230000006037 cell lysis Effects 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 108091092356 cellular DNA Proteins 0.000 description 1
- 238000011210 chromatographic step Methods 0.000 description 1
- 239000007979 citrate buffer Substances 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 238000004737 colorimetric analysis Methods 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 230000022811 deglycosylation Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 230000000994 depressogenic effect Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000006471 dimerization reaction Methods 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 239000013613 expression plasmid Substances 0.000 description 1
- 239000013604 expression vector Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 210000003000 inclusion body Anatomy 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 239000000411 inducer Substances 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 239000000543 intermediate Substances 0.000 description 1
- 239000013067 intermediate product Substances 0.000 description 1
- 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 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000008774 maternal effect Effects 0.000 description 1
- 230000002906 microbiologic effect Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 231100000590 oncogenic Toxicity 0.000 description 1
- 230000002246 oncogenic effect Effects 0.000 description 1
- 125000000636 p-nitrophenyl group Chemical group [H]C1=C([H])C(=C([H])C([H])=C1*)[N+]([O-])=O 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- 229920002704 polyhistidine Polymers 0.000 description 1
- 238000001742 protein purification Methods 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 230000010076 replication Effects 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 239000007974 sodium acetate buffer Substances 0.000 description 1
- 229910000162 sodium phosphate Inorganic materials 0.000 description 1
- 239000008174 sterile solution Substances 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 101150006154 strH gene Proteins 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 230000009897 systematic effect Effects 0.000 description 1
- 101150093706 tor gene Proteins 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- 238000000870 ultraviolet spectroscopy Methods 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
Classifications
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
- B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
- B01D15/32—Bonded phase chromatography
- B01D15/325—Reversed phase
- B01D15/327—Reversed phase with hydrophobic interaction
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
- B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
- B01D15/34—Size selective separation, e.g. size exclusion chromatography, gel filtration, permeation
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
- B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
- B01D15/36—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving ionic interaction
- B01D15/361—Ion-exchange
- B01D15/363—Anion-exchange
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
- B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
- B01D15/38—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving specific interaction not covered by one or more of groups B01D15/265 - B01D15/36
- B01D15/3804—Affinity chromatography
- B01D15/3828—Ligand exchange chromatography, e.g. complexation, chelation or metal interaction chromatography
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/16—Extraction; Separation; Purification by chromatography
Definitions
- the invention relates to the field of biotechnology, and in particular to methods for the preparation and production of therapeutic preparations, isolation and purification of recombinant proteins, in particular in the preparation of a highly purified recombinant exo-beta-N-acetylglucosaminidase StrH (b-N-GlcNAcase StrH) preparation.
- b-N-GlcNAcase StrH exo-beta-N-acetylglucosaminidase StrH
- the main areas of application of the bacterial b-N-GlcNAcase StrH obtained by the proposed method are its inclusion in the technological process for obtaining a number of medical drugs (LS), as well as biomedical research in the field of glycobiology.
- the invention solves the problem of creating a method for producing large quantities of highly purified enzymatically active recombinant b-N-GlcNAcase StrH, suitable for use at the remodeling stage to obtain therapeutically functional preparations of glycoproteins with desired properties, to develop tools for highly sensitive analysis of glycan chains of glycoproteins of tissues of plants, animals and eukaryotic microorganisms.
- the invention also relates to a method for producing a recombinant b-N-GlcNAcase StrH preparation having a higher degree of purity compared to commercial preparations, homogeneous in the target product, with a lower content of endotoxins and producer DNA in comparison with commercial preparations.
- FIG. 1 The structure of the N-glycosylated human glycoprotein.
- the names of the sugar residues are located under their respective symbols.
- the lines are the bonds between sugar residues, the numbers above are the specificity of the bond.
- Arrows are bonds that are potentially cleaved by glycosidases, indicated at the top of the diagram;
- FIG. 1 Substrate specificity of b-N-GlcNAcase StrH. Data for two constructs: 1 - corresponds to the full-size StrH, 2 - catalytic domain GH20A. The complexity of glycans increases from (a) to (e). Activity on all of the substrates presented is observed only for the full-sized construct. For GH20A, reduced activity on the substrate (c) and its absence on substrates (d) or (e) are observed.
- FIG. 3 Map of plasmid pMT1618 (expression vector based on pET15 for obtaining streptococcal b-N-GlcNAcase StrH). Designations: T7 promoter - promoter of T7 RNA polymerase; N-terminal His-tagged bN-GlcNAcase StrH is a protein gene of His-tagged (N-terminus) bN-GlcNAcase StrH, a component of which is a sequence of 20 amino acid residues containing a hexahistidine tag, and 1117 amino acid residues (without a signal peptide, one of two G5 domains and an anchor peptide) b-N-GlcNAcase StrH Streptococcus pneumoniae; T7 terminator - terminator of T7 RNA polymerase; AmpR promoter - promoter of the ampicillin resistance gene; AmpR - ampicillin resistance gene; ori - origin of replication; lad - lactose repressor gene;
- FIG. 4 Dynamics of biomass accumulation during cultivation of the producer strain E. coli VKPM B-12747 in a volume of 15 l. Abscissa - cultivation hours, along the ordinate axis - biomass accumulation;
- FIG. 5 Elution profile of the target b-N-GlcNAcase StrH protein - gel filtration on Superdex 200.
- FIG. 6 Result of Isolation of b-N-GlcNAcase StrH. PAGE analysis by electrophoresis. Comparison of a commercial preparation and the resulting recombinant b-N-GlcNAcase StrH preparation. Lane 1
- FIG. 7 The result of the analysis of GF HPLC. Industrial Series b-N- GlcNAcase StrH.
- FIG. 8 The result of the analysis of GF HPLC. Comparison of a commercial preparation and a recombinant b-N-GlcNAcase StrH preparation with enhanced chromatographic purity.
- glycosidases are of limited importance, since they are active only at low pH, or only on low molecular weight substrates, and, as a rule, there are difficulties in obtaining pure preparations in the production of drugs from natural sources. Usually they are contaminated with impurities of other glycosidases, proteases, bacterial endotoxins. In addition, a limited number of exo-acetylglucosaminidases, which are isolated and produced from natural sources, are commercially available. The list is presented in Table 1. (4. US Patent 7094563 B2, Isolation and composition of novel glycosidases // 2006.).
- b-N-GlcNAcase StrH Diplococcus pneumonia isolated from a natural source was described in R. Colin Hughes at al., The Extracellular Glycosidases of Diplococcus pneumoniae. Purification and Properties of a b-N-Acetylglucosaminidase. Action on a Derivative of the a- Acid Glycoprotein of Human Plasma, // Biochemistry - 1964.-V.3.-N.10.-P.1543-1548. It was found that b-N-GlcNAcase StrH is active both at neutral pH and on low and high molecular weight substrates.
- the commercially available sorbent glutathione (g-glutamylcysteinylglycine) Sepharose may be affected by g-glutamyl transpeptidase found in untreated cell lysates. Therefore, glutathione sorbent has a finite life and can be regenerated and reused 4-20 times, unlike, for example, IMAC sorbents, which can be used almost indefinitely (Michelle E. Kimple at al., Overview of Affinity Tags for Protein Purification , HHS Public Access Author manuscript, // Curr Protoc Protein Sci.- 2015- V.73.-P. 1-26).
- Chaperonin - 70 kDa E. coli (heat shock protein) is also adsorbed onto the sorbent and coelutes with the target GST fusion protein.
- Closest to the claimed invention can be considered a method for cleaning bN-GlcNAcase StrH, disclosed in Benjamin Pluvinage at al, Conformational Analysis of StrH, the Surface-Attached exo-b-dN-Acetylglucosaminidase from Streptococcus pneumoniae, // Journal of Molecular Biology- 2013 .- V. 425.- N. 23. -P. 334-349.
- This method involves the purification of a homologous variant of the enzyme and its variants in the form of a fusion protein with a polyhistidine sequence, in analytical quantities.
- the target proteins were purified by metal chelate chromatography on an IMAC Sepharose, followed by thrombin hydrolysis and anion exchange chromatography on UNOsphere Q (Bio-Rad Laboratories), gel filtration on Sephacryl S200 (GE Biosciences), followed by concentration by ultrafiltration using a 10 kDa cut-off membrane.
- This purification scheme using thrombin is unacceptable for large-scale production of the drug due to the already mentioned reasons for the hydrolysis of factor Xa.
- the authors did not report the level of bacterial endotoxins in the preparation. The removal of bacterial endotoxins is a fundamental and necessary requirement, both from the point of view of Russian and international standards (GF XIII, Document Q AS / 11.452 FINAL July 2012).
- the inventors propose a new method for producing recombinant bN-GlcNAcase StrH from Streptococcus pneumoniae, including the synthesis of a DNA sequence optimized for translation into E.
- the main technical result of the invention is the production on an industrial scale highly purified active drug b-N-GlcNAcase StrH, suitable for use in the manufacture of drugs and glycobiological studies.
- a strain of a genetically modified microorganism E.coli VKPM B-12747 was created: the strain was obtained by transforming the recipient host strain BL21 (DE3) with plasmids pLysS carrying the T7 lysozyme gene, which is an inhibitor of T7 RNA polymerase, pMT1618 (pET15b-STRH_STH_STH_ ), which carries the bN-GlcNAcase StrH protein gene, of which a sequence of 20 amino acid residues containing a hexahistidine tag, a thrombin recognition site and 1117 amino acid residues (without a signal peptide, one of two G5 domains and anchor peptide tida) b-N-GlcNAcase StrH (Fig.Z.).
- Refolding of the product is not required, since it is synthesized in the bacterial cytoplasm mainly in the dissolved state during the cultivation of E. coli VKPM B-12747 with the level of biosynthesis of variants of the target protein of at least 40 mg / l of culture fluid.
- chromatographic purification scheme which includes: metal chelate chromatography on IMAC Sepharose 6 FF, chromatography in slip mode on Blue Sepharose FF, anion exchange chromatography in Toyopearl Giga Cap Q-65 OM, chromatography in slip mode on Butyl Capto, concentration anion exchange chromatography on Q Sepharose FF.
- concentration by ultrafiltration using a membrane with a cut-off of 50 kDa and gel filtration on Superdex 200 can be additionally included in the scheme.
- the introduction to the purification scheme of the Blue Sepharose chromatography step in a slip mode allows one to significantly purify the target protein from contaminating impurities of cellular proteins coeluting with the target protein at all stages.
- TOYOPEARL GigaCap Q-650M a high-resolution, high molecular weight anion-exchange sorbent optimized for the capture and purification of high molecular weight proteins.
- the conditions of planting and removal of the target protein were selected, which allow maximum purification of the target protein from contaminating impurities of cellular proteins and lipopolysaccharides.
- the method differs from previously known also by the use of Butyl Capto chromatography in slip mode, which allows removing contaminants of hydrophobic proteins and lipopolysaccharides.
- HECs genetic engineering constructs
- pMT1618 For the expression of bN-GlcNAcase StrH, genetic engineering constructs (HECs) of pMT1618 are obtained.
- GICs a vector-based cloning scheme was developed pET-15b (Novagen), which assumes the receipt of the full version having 6xHis-tag at the N-terminus.
- TOP Gene Technologies ordered a synthetic gene encoding bN-GlcNAcase StrH (the genomic sequence of the bacterium Streptococcus pneumoniae encoding 1117 amino acid residues (without signal peptide, one of two G5 domains and anchor peptide) b-N-acetylglucosaminidase ( strH), fused at the 5 'end with part of the vector sequence of the maternal plasmid pET15b (+) encoding 20 amino acid residues containing hexahistidine tag) flanked by Ndel restriction sites, BamHI, containing internal restriction sites and Nhel to obtain a "catalytic" domain and stop codons at the 3'-end of the sequence (TGATAA).
- bN-GlcNAcase StrH the genomic sequence of the bacterium Streptococcus pneumoniae encoding 1117 amino acid residues (without signal peptide, one of two G5 domains and anchor peptide) b-N-acetyl
- codon optimization was performed for protein expression in E. coli, taking into account the requirement that there are no Ndel, BamHI, Xhol, and Nhel restriction endonuclease sites in the sequence. Codon optimization of the obtained bN-GlcNAcase StrH sequence was verified at http://gcua.schoedl.de/sequential v2.html. The correspondence of the translated amino acid sequence to the ordered one was confirmed by alignment in the MEGA 6.0 program. The absence of unwanted restriction sites was confirmed using CloneManager 9.0 software.
- the synthesis and restriction mapping of the synthetic gene pAPGl lO-bN-GlcNAcase StrH was performed.
- the fragment encoding streptococcal bN-GlcNAcase StrH from plasmid pAPGHO-STRH STRPN was cloned into the vector plasmid pET15b at the Ndel, BamHI restriction sites. Received during restriction fragments were purified and used for ligation. Chemically competent E. coli XL10-GOLD cells were transformed with a ligase mixture. Cells were seeded to obtain transformed clones.
- Plasmid DNA was then isolated and restriction mapping using Pstl restriction endonuclease was performed. A plasmid DNA sample was transferred for sequencing to Eurogen. The resulting sequence was aligned with the "theoretical" sequence pMT 1618.
- plasmid DNA GIC pMT1618 was transformed into strains BL21 (DE3) pLysS.
- the super-producing clones of b-N-GlcNAcase StrH were selected based on analytical expression.
- the strain was deposited in the All-Russian collection of industrial microorganisms (VKPM) under inventory number - VKPM B-12747.
- the stage of biosynthesis of the hybrid protein begins.
- the expression of bN-GlcNAcase StrH protein is under the control of the promoter, which is depressed by introducing a solution of IPTG to a final concentration of 0.0476 wt.%.
- the level of biosynthesis of the variants of the target bN-GlcNAcase StrH protein is at least 15 mg / l of culture fluid.
- Bacterial biomass was collected by centrifugation and stored at - 70 ° C. Cellular biomass was lysed, the lysate was clarified by centrifugation, and bN-GlcNAcase StrH protein was isolated from the lysate by binding to the target protein by column chelate chromatography IMAC Sepharose FF. The enzyme was further purified by sequential chromatography on Blue Sepharose FF in slip mode, anion exchange Toyopearl Giga Cap Q-650M, Butyl Capto in slip mode, chromatography on Q HP, concentration and gel filtration. At each stage, the degree of purification is monitored by electrophoresis in PAAT.
- the protein yield is at least 0.04 mg per liter of bacterial culture.
- the final product has the following parameters:
- the specific activity of the b-N-GlcNAcase StrH preparations of the present invention is from 230 units / mg protein and above.
- the GlcNAcase StrH of the present invention is not more than 4 UE / mg protein.
- the residual DNA of the producer strain in the b-N-GlcNAcase StrH preparation of the present invention is preferably not more than 4.0 pg / mg protein.
- the preparation of bN-GlcNAcase StrH based on an optimized synthetic DNA sequence with a short sequence of six histidines at the N-terminus of the recombinant protein occurs without removing the hexahistidine tag, which simplifies the purification and removes the use of expensive tag removers from the purification scheme.
- the amino acid sequence of bN-GlcNAcase StrH is represented by SEQ ID NO 2.
- the inventors use IMAC Sepharose 6 FF metal chelate chromatography to completely sorb the target protein with a hexahistidine tag. The use at this stage of washing with acidic buffer with high ionic strength can significantly remove impurities of cellular proteins from the target product.
- chromatography on Blue Sepharose in slip mode allows you to clean the target protein from contaminating impurities of cellular proteins coeluting with the target protein at all stages.
- Anion-exchange chromatography is carried out on Toyopearl Giga Cap Q-650M, a high-resolution, high-molecular-weight anion-exchange sorbent optimized for the capture and purification of proteins with high molecular weight.
- the conditions for planting and removing the target protein are selected in such a way as to maximize purification of the target protein from contaminating impurities of cellular proteins and bacterial endotoxins.
- Breakthrough chromatography on Butyl Capto removes contaminating contaminants of hydrophobic proteins and bacterial endotoxins.
- the specific activity of b-N-GlcNAcase StrH preparations obtained by the method disclosed in the invention is 240 units / mg protein; the content of bacterial endotoxins is from 2 U / mg to 4 U / mg protein; the residual DNA content of the producer strain is not more than 4.0 pg / mg protein.
- the claimed method allows to obtain a preparation of recombinant bN-GlcNAcase StrH, having a higher degree of purity compared with commercial analogues, the comparison results are presented in Table 2.
- the claimed method is implemented for the producer strain E. coli VKPM B-12747 when cultured in a fermenter (Biotron LiFlus SP / SL-20L, South Korea) in a volume of nutrient medium of 15 l. pH is maintained at a level of 7, 0-7, 6. Cultivation is carried out at a temperature of 37 ° C. The relative content of dissolved oxygen is maintained at a level of 20-100% by mixing at a speed 800 rpm and air supply with a volume flow of 15 l / min. Inoculum in a volume of 0.75 l, grown to an optical density measured at a wavelength of 600 nm (OD600) equal to 1.5 p.u. (optical units), contribute to the fermenter with a sterile culture medium.
- a fermenter Biotron LiFlus SP / SL-20L, South Korea
- the main parameters of culture growth are monitored: OD600, pH, cell morphology, relative dissolved oxygen content.
- an inducer is introduced - a sterile solution of isopropyl-b-O-1 - thiogalactopyranoside to a final concentration of 0.0476 wt.% (2 mm).
- Cultivation is carried out for 5-6 hours.
- the final optical density is 28.2 pu, the biomass yield is 44 g / l.
- the productivity of the target product of E. coli strain VKPM B-12747 is 40 mg / l of culture fluid.
- Figure 4. The dynamics of biomass accumulation during cultivation of the producer strain E. coli VKPM B-12747 is presented.
- Example 2 Isolation and purification of b-N-GlcNAcase StrH protein from bacterial biomass. Obtaining an industrial series of the drug b-N-GlcNAcase StrH.
- Buffer A 20 mM Tris-HCl, 0.5 M NaCl, pH 7.8;
- Frozen cell biomass of 180 g, 60 g of ZXR were suspended in 350 ml of buffer A with PMSF up to 0.003% for 45 min on ice and destroyed in a Panda Plus 2000 high-pressure flow homogenizer (GEA Niro Soavi).
- the homogenizer is washed with 250 ml of purified water cooled to 0 ° C, adjusted to buffer A cooled to 0 ° C, setting of stage II is 140 bar, setting of stage I is 800 bar, then lyse the cell suspension in this mode, collecting the lysate in a container placed in an ice bath, rinse the homogenizer with 30 ml of buffer A.
- the lysis is repeated 2 more times in the same mode.
- a 1500 ml lysate is clarified by centrifugation at 33250 x g (JLA-16.250) at 4 ° C for 60 minutes.
- the supernatant is 1500 ml, diluted with buffer A to 1800 ml, adjusted to pH 7.8 with 2M NaOH, filtered through a Sartopor 2 150 depth filter and applied to a Ni IMAC Sepharose column equilibrated with buffer A. After application, the column is washed with buffer A and sodium acetate buffer with 1 M NaCl, pH 5.0. The target protein is eluted with 250 mM imidazole.
- the breakthrough with Blue Sepharose 6 FF is diluted with purified water to 4 ° C purified to a conductivity of 1.8 mSm / sm, adjusted to pH 6.5 and applied to a Toyopearl Giga Cap Q-650M column equilibrated with 50 mM MES buffer, pH 6.5 .
- the target protein is eluted with a gradient from 3MM to 0.3 M NaCl in the start buffer.
- the eluate with Toyopearl Giga Cap Q-65 OM is diluted with purified water to a conductivity of 2.04 mSm / sm, adjusted to pH 7.5 and applied to columns with Q Sepharose FF with HiTrap Butyl Capto pre-column, equilibrated with 20 mM Tris-HO buffer, pH 7.5. Washed with starting buffer until the adsorbed components were eluated, the HiTrap Butyl Capto column was disconnected, washed with buffer B (20 mM Tris-HCl, ImM EDTA, pH 7.5) and eluted with 250 mM NaCl in buffer B.
- the eluate with Q Sepharose FF was diluted with buffer B to the finished form and sterilely filtered through FILTER-MAX, “rapid” - 150 ml, 0.22 pm, aliquoted and stored at minus 20 ° C.
- the content of bacterial endotoxins From 9.6 to 19.2 EU / ml.
- the purity of the sample by GF HPLC is: 90.0%.
- Residual DNA of producer strain (E. colli): ⁇ 4.0 pg / mg.
- Example 3 Obtaining protein b-N-GlcNAcase StrH with improved quality characteristics.
- Concentrated anion exchange chromatography fractions containing the target protein were combined and concentrated on Amicon Ultrac 50 Centricons. Then, the concentrate was applied to a Superdex 200 column using 20 mM Tris HCl, 0.05 M NaCl buffer as a mobile phase, ImM EDTA pH 7.5. The elution of the target protein is carried out in the same buffer. The elution profile is shown in FIG. 5.
- the eluted target bN-GlcNAcase StrH protein is diluted with the mobile phase buffer to a protein concentration of not more than 1 mg / ml and, observing aseptic conditions, sterilely filtered through a 0.22 ⁇ m filter, Packed in aliquots and stored at - 20 ° C.
- Determination of the activity of the recombinant b-N-GlcNAcase StrH is carried out by staging a test on a synthetic NAG substrate (4-Nitrophenyl N-acetyl-P-D-glucosaminide) using the “b-N-Acetylglucosaminidase Assay Kit” (Sigma). Hydrolysis of the NAG substrate (4-Nitrophenyl N-acetyl ⁇ -D-glucosaminide) by the b-N-Acetylglucosaminidase enzyme produces the product p-nitrophenol, the content of which is measured colorimetrically at a wavelength of 405 nm.
- One unit of activity (1 unit) should provide hydrolysis of 1.0 pmole of 4-Nitrophenyl N-acetyl ⁇ -D-glucosaminide substrate to produce p-nitrophenol and N-acetyl ⁇ -D-glucosaminide products in 1 minute at pH 4.7 and a temperature of 37 ° C .
- prepare a solution of the substrate "4-Nitrophenyl N-acetyl ⁇ -D-glucosaminide" with a concentration of 1 mg / ml To do this, dissolve 10 mg of the substrate in 10 ml of a 0.09M Citrate Buffer Solution solution.
- a b-N-Acetylglucosaminidase from Jack beans solution is prepared by diluting 100 times in a Dilution Buffer solution.
- Prepare a Stop Solution by diluting the contents of the vial in 118 ml of purified water.
- dilutions of b-N-Acetylglucosaminidase (NAG) were prepared 50, 100, 200, 400 times in Dilution Buffer.
- the optical density of the solutions is measured at a wavelength of 405 nm.
- the enzyme activity is calculated by the formula:
- Electrophoresis is carried out in a 4-20% gradient PAGE under reducing conditions, stained with silver salts (Thermo Scientific).
- Sample preparation of samples is carried out by diluting the samples to a concentration of ⁇ 50 ⁇ g / ml by adding a buffer for applying samples (Sb) with the addition of a 2 M solution of dithiothreitol (DTT). Warm up for 5 minutes at (100 ⁇ 2) OS. Centrifuged for 1 min., 13000 rpm and apply samples (solutions obtained by diluting the samples) 10 ⁇ l per well of gel, molecular weight markers - 5 ⁇ l per well of gel. Electrophoresis is carried out at a voltage of 400 V, current strength of 30 mA, the time until the dye is released. Then stained with silver salts according to the instructions for the Thermo Scientific kit. Results are presented on
- Example 6 Testing purified preparations of recombinant b-N-GlcNAcase StrH in comparison with the commercial preparation of b-N-GlcNAcase Case for the content of bacterial endotoxins.
- Example 7 Testing the chromatographic purity of purified preparations of recombinant b-N-GlcNAcase StrH in comparison with the commercial preparation b-N-GIcNAcase.
- Example 8 Testing purified preparations of recombinant b-N-GlcNAcase StrH in comparison with the commercial preparation of b-N-GlcNAcase Strain on residual DNA of the producer strain.
- Testing for the residual DNA of the producer strain is carried out by real-time quantitative PCR, not more than 20 pg / mg.
- QPCR quantitative polymerase chain reaction
- the Taqman system was chosen, in which, upon propagation of a specific DNA sequence, the probe is degraded by DNA polymerase.
- a set of primers and a probe for detecting the DNA of the producer E. coli strain are shown in Table 6.
- the samples were purified using the QIAamp DNA Blood Mini Kit (Qiagen). The final volume during elution was 50 ⁇ l. 10 ⁇ l of sample was added to the qPCR reaction (sample). The number of repetitions for each sample is 3.
- the amount of DNA in the sample is less than 0.5 pg below the limit of reliable detection.
- Table 7 The results of the analysis are presented in Table 7.
- Table 6. A set of primers and probe for the detection of DNA of a producer strain of E. coli.
Landscapes
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Organic Chemistry (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
The invention relates to biotechnology, in particular to methods for producing and manufacturing therapeutic preparations and separating and purifying recombinant proteins. An industrial method is disclosed for producing a biologically active preparation of recombinant exo-beta-N-acetylglucosaminidase StrH (β-N-GlcNAcase StrH), produced in accordance with GMP regulations and suitable for use at the remodeling stage in the production of therapeutically functional preparations of glycoproteins with specific properties, as well as for the development of instruments for the highly sensitive analysis of glycan chains of glycoproteins of plant and animal tissues and eukaryotic microorganisms.
Description
СПОСОБ ПОЛУЧЕНИЯ РЕКОМБИНАНТНОЙ METHOD FOR PRODUCING RECOMBINANT
БЕТА-1Ч-АЦЕТИЛГЛЮКОЗАМИНИДАЗЫ STRH ИЗ Beta-1H-Acetylglucosaminidase STRH
STREPTOCOCCUS PNEUMONIAE STREPTOCOCCUS PNEUMONIAE
Изобретение относится к области биотехнологии, а именно к способам получения и производства терапевтических препаратов, выделения и очистки рекомбинантных белков, в частности в получении высокоочищенного препарата рекомбинантной экзо-бета-N- ацетилглюкозаминидазы StrH (b-N-GlcNAcase StrH). Основными сферами применения полученной предлагаемым способом бактериальной b-N-GlcNAcase StrH является включение её в технологический процесс получения ряда медицинских лекарственных препаратов (ЛС), а также биомедицинские исследования в области гликобиологии. The invention relates to the field of biotechnology, and in particular to methods for the preparation and production of therapeutic preparations, isolation and purification of recombinant proteins, in particular in the preparation of a highly purified recombinant exo-beta-N-acetylglucosaminidase StrH (b-N-GlcNAcase StrH) preparation. The main areas of application of the bacterial b-N-GlcNAcase StrH obtained by the proposed method are its inclusion in the technological process for obtaining a number of medical drugs (LS), as well as biomedical research in the field of glycobiology.
Изобретение решает задачу создания способа получения больших количеств высокоочищенной ферментативно активной рекомбинантной b-N-GlcNAcase StrH, пригодной для использования на стадии ремоделирования для получения терапевтически функциональных препаратов гликопротеинов с заданными свойствами, для разработки инструментов для высокочувствительного анализа гликановых цепей гликопротеинов тканей растений, животных и эукариотических микроорганизмов. Изобретение относится также к способу получения препарата рекомбинантной b-N-GlcNAcase StrH, имеющего более высокую степень чистоты по сравнению с коммерческими препаратами, гомогенного по целевому продукту, с более низким содержанием эндотоксинов и ДНК продуцента в сравнении с коммерческими препаратами. The invention solves the problem of creating a method for producing large quantities of highly purified enzymatically active recombinant b-N-GlcNAcase StrH, suitable for use at the remodeling stage to obtain therapeutically functional preparations of glycoproteins with desired properties, to develop tools for highly sensitive analysis of glycan chains of glycoproteins of tissues of plants, animals and eukaryotic microorganisms. The invention also relates to a method for producing a recombinant b-N-GlcNAcase StrH preparation having a higher degree of purity compared to commercial preparations, homogeneous in the target product, with a lower content of endotoxins and producer DNA in comparison with commercial preparations.
ОПИСАНИЕ ЧЕРТЕЖЕЙ DESCRIPTION OF DRAWINGS
Фиг. 1. Схема строения N-гликозилированного человеческого
гликопротеина. Названия сахарных остатков расположены под соответствующими им символами. Линии - связи между сахарными остатками, числа наверху - специфичность связи. Стрелки - связи, которые потенциально расщепляются гликозидазами, обозначенными вверху схемы; FIG. 1. The structure of the N-glycosylated human glycoprotein. The names of the sugar residues are located under their respective symbols. The lines are the bonds between sugar residues, the numbers above are the specificity of the bond. Arrows are bonds that are potentially cleaved by glycosidases, indicated at the top of the diagram;
Фиг.2. Субстратная специфичность b-N-GlcNAcase StrH. Данные для двух конструктов: 1 - соответствует полноразмерному StrH, 2 - каталитический домен GH20A. Сложность гликанов возрастает от (а) к (е). Активность на всех представленных субстратах наблюдается только для полноразмерного конструкта. Для GH20A наблюдается пониженная активность на субстрате (с) и ее отсутствие на субстратах (d) или (е). Figure 2. Substrate specificity of b-N-GlcNAcase StrH. Data for two constructs: 1 - corresponds to the full-size StrH, 2 - catalytic domain GH20A. The complexity of glycans increases from (a) to (e). Activity on all of the substrates presented is observed only for the full-sized construct. For GH20A, reduced activity on the substrate (c) and its absence on substrates (d) or (e) are observed.
Фиг. 3. Карта плазмиды рМТ1618 (экспрессионный вектор на основе рЕТ15Ь для получения стрептококковой b-N-GlcNAcase StrH). Обозначения: Т7 promoter - промотор Т7 РНК-полимеразы; N-terminal His-tagged b-N-GlcNAcase StrH - ген белка His-тэгированного (N-конец) b-N-GlcNAcase StrH, составной частью которого являются последовательности 20 аминокислотных остатков, содержащих гексагистидиновый тэг, и 1117 аминокислотных остатков (без сигнального пептида, одного из двух G5 доменов и якорного пептида) b- N-GlcNAcase StrH Streptococcus pneumoniae; T7 terminator - терминатор T7 РНК-полимеразы; AmpR promoter - промотер гена устойчивости к ампициллину; AmpR - ген устойчивости к ампициллину; ori - ориджин репликации; lad - ген лактозного репрессора; lad promoter - промотор гена лактозного репрессора; Ndel - сайт узнавания эндонуклеазы рестрикции Ndel; BamHI - сайт узнавания эндонуклеазы рестрикции ВатШ FIG. 3. Map of plasmid pMT1618 (expression vector based on pET15 for obtaining streptococcal b-N-GlcNAcase StrH). Designations: T7 promoter - promoter of T7 RNA polymerase; N-terminal His-tagged bN-GlcNAcase StrH is a protein gene of His-tagged (N-terminus) bN-GlcNAcase StrH, a component of which is a sequence of 20 amino acid residues containing a hexahistidine tag, and 1117 amino acid residues (without a signal peptide, one of two G5 domains and an anchor peptide) b-N-GlcNAcase StrH Streptococcus pneumoniae; T7 terminator - terminator of T7 RNA polymerase; AmpR promoter - promoter of the ampicillin resistance gene; AmpR - ampicillin resistance gene; ori - origin of replication; lad - lactose repressor gene; lad promoter - promoter of the lactose repressor gene; Ndel — Ndel restriction endonuclease recognition site; BamHI - recognition site for restriction endonuclease
Фиг. 4. Динамика накопления биомассы при культивировании штамма- продуцента E.coli ВКПМ В- 12747 в объеме 15 л. По оси абсцисс
- часы культивирования, по оси ординат - накопление биомассы;FIG. 4. Dynamics of biomass accumulation during cultivation of the producer strain E. coli VKPM B-12747 in a volume of 15 l. Abscissa - cultivation hours, along the ordinate axis - biomass accumulation;
Фиг. 5. Профиль элюции целевого белка b-N-GlcNAcase StrH - гель- фильтрация на Superdex 200. FIG. 5. Elution profile of the target b-N-GlcNAcase StrH protein - gel filtration on Superdex 200.
Фиг. 6. Результат выделения b-N-GlcNAcase StrH. Анализ электрофорезом в ПААГ. Сравнение коммерческого препарата и полученного препарата рекомбинантной b-N-GlcNAcase StrH. Дорожка 1 FIG. 6. Result of Isolation of b-N-GlcNAcase StrH. PAGE analysis by electrophoresis. Comparison of a commercial preparation and the resulting recombinant b-N-GlcNAcase StrH preparation. Lane 1
- маркеры молекулярной массы Thermo Scientific; Дорожка 2 - рекомбинантная коммерческая b-N-ацетилглюкозаминидаза; Дорожка 3 - Очищенный препарат рекомбинантной b-N-GlcNAcase StrH. - molecular weight markers Thermo Scientific; Lane 2 - recombinant commercial b-N-acetylglucosaminidase; Lane 3 - Purified recombinant b-N-GlcNAcase StrH preparation.
Фиг. 7. Результат анализа ГФ ВЭЖХ. Промышленная серия b-N- GlcNAcase StrH. FIG. 7. The result of the analysis of GF HPLC. Industrial Series b-N- GlcNAcase StrH.
Фиг. 8. Результат анализа ГФ ВЭЖХ. Сравнение коммерческого препарата и препарата рекомбинантной b-N-GlcNAcase StrH с повышенной хроматографической чистотой. FIG. 8. The result of the analysis of GF HPLC. Comparison of a commercial preparation and a recombinant b-N-GlcNAcase StrH preparation with enhanced chromatographic purity.
Отсутствие отечественных коммерчески доступных препаратов рекомбинантной b-N-GlcNAcase StrH из Streptococcus pneumoniae, экспрессированных в E.coli, и чрезвычайно высокая стоимость препаратов у зарубежных производителей таких, как QA-Bio, Amsbio, ProZyme, Sigma делает коммерчески невыгодным использование этих препаратов при производстве лекарственных препаратов. Препараты данных фирм производятся не в соответствии со стандартами «Надлежащей производственной npaicraKH»/«Good Manufacturing Practice» (GMP) и предназначены только для аналитических исследований. The lack of domestic commercially available recombinant bN-GlcNAcase StrH preparations from Streptococcus pneumoniae expressed in E. coli, and the extremely high cost of drugs from foreign manufacturers such as QA-Bio, Amsbio, ProZyme, Sigma make the use of these drugs commercially unprofitable in the manufacture of drugs . The products of these companies are not manufactured in accordance with the standards of Good Manufacturing npaicraKH / Good Manufacturing Practice (GMP) and are intended only for analytical studies.
Подробные характеристики b-N-GlcNAcase StrH, принадлежащей к большому семейству 20 гликозидаз - гидролаз, которые гидролизуют терминальный сахарный остаток с нередуцирующего конца олигосахаридов и углеводной цепи гликоконьюгатов, таких как
гликопротеины и гликолипиды ( Фиг.1.), даны в публикациях Kobata Akira, Use of Endo- and Exoglycosidases for Structural Studies of Glycoconiugates, //Analytical biochemistry - 1979.- V.100.-P.1-14; Intra J at al., Phylogenetics analyses multiple changes of substrate specificity the glycosil hydrolase 20 family, //BMC Evol. Biol. -2008.- P. 8214;.Lowrie R.Glascow at al, Systematic Purification of Five Glycosidases from Streptococcus ( Diplococcus) pneumoniae, // The Journal of Biological Chemistry - 1977. -V.252.- N. 23.- Issue of December 10.- P. 8615-8623. Раскрыто большое количество экзо- ацетилглюкозаминидаз, выделенных из множества организмов, таких как бактерии, вирусы, растительные и животные ткани, которые широко используются, как инструмент анализа структуры и функций гликоконьюгатов, олигосахаридов, ассоциированных с гликопротеинами и клеточными мембранами, и пользуются большим спросом в гликобиологических исследованиях и промышленных процессах. Однако все они обладают разной субстрат- специфичностью и многие из описанных гликозидаз имеют ограниченное значение, так как активны только при низких pH, или только на низкомолекулярных субстратах, и, как правило, при производстве препаратов из природных источников существуют трудности с получением чистых препаратов. Обычно они загрязнены примесями других гликозидаз, протеаз, бактериальных эндотоксинов. Кроме того, ограниченное количество экзо- ацетилглюкозаминидаз, которые выделяются и производятся из природных источников, коммерчески доступно. Перечень представлен в Таблице 1. (4. US Patent 7094563 В2, Isolation and composition of novel glycosidases// 2006.). Detailed characteristics of bN-GlcNAcase StrH, belonging to the large family of 20 glycosidases - hydrolases that hydrolyze the terminal sugar residue from the non-reducing end of the oligosaccharides and the carbohydrate chain of glycoconjugates, such as glycoproteins and glycolipids (Figure 1.) are given in the publications Kobata Akira, Use of Endo- and Exoglycosidases for Structural Studies of Glycoconiugates, // Analytical biochemistry - 1979.- V.100.-P.1-14; Intra J at al., Phylogenetics analyses multiple changes of substrate specificity the glycosil hydrolase 20 family, // BMC Evol. Biol. -2008.- P. 8214; .Lowrie R. Glascow at al, Systematic Purification of Five Glycosidases from Streptococcus (Diplococcus) pneumoniae, // The Journal of Biological Chemistry - 1977. -V.252.- N. 23.- Issue of December 10.- P. 8615-8623. A large number of exo-acetylglucosaminidases isolated from many organisms, such as bacteria, viruses, plant and animal tissues, are widely used as a tool for analyzing the structure and functions of glycoconjugates, oligosaccharides associated with glycoproteins and cell membranes, and are in great demand in glycobiological research and industrial processes. However, they all have different substrate specificity and many of the glycosidases described are of limited importance, since they are active only at low pH, or only on low molecular weight substrates, and, as a rule, there are difficulties in obtaining pure preparations in the production of drugs from natural sources. Usually they are contaminated with impurities of other glycosidases, proteases, bacterial endotoxins. In addition, a limited number of exo-acetylglucosaminidases, which are isolated and produced from natural sources, are commercially available. The list is presented in Table 1. (4. US Patent 7094563 B2, Isolation and composition of novel glycosidases // 2006.).
Таблица 1. Коммерчески доступные экзо-р-К-ацетилглюкозаминидазы из природных источников
Table 1. Commercially available exo-p-K-acetylglucosaminidases from natural sources
Впервые b-N-GlcNAcase StrH Diplococcus pneumonia, выделенная из природного источника, охарактеризована в публикации R. Colin Hughes at al., The Extracellular Glycosidases of Diplococcus pneumoniae. Purification and Properties of a b-N-Acetylglucosaminidase. Action on a Derivative of the a- Acid Glycoprotein of Human Plasma, //Biochemistry - 1964.-V.3.-N.10.-P.1543-1548. Было выяснено, что b-N-GlcNAcase StrH активна и при нейтральной pH и на низко- и высокомолекулярных субстратах. For the first time, b-N-GlcNAcase StrH Diplococcus pneumonia isolated from a natural source was described in R. Colin Hughes at al., The Extracellular Glycosidases of Diplococcus pneumoniae. Purification and Properties of a b-N-Acetylglucosaminidase. Action on a Derivative of the a- Acid Glycoprotein of Human Plasma, // Biochemistry - 1964.-V.3.-N.10.-P.1543-1548. It was found that b-N-GlcNAcase StrH is active both at neutral pH and on low and high molecular weight substrates.
В публикации Valerie A. Clarke at al., Cloning and Expression of the b- N-Acetylglucosaminidase Gene from Streptococcus pneumoniae. Generation of truncated enzymes with modified aglycon specificity, // The Journal of Biological Chemistry - 1995.-V.270.-N.15.- P. 8805-8814), впервые идентифицирована структура гена b-N-GlcNAcase StrH из Streptococcus pneumoniae. Показано, что экспрессия почти полного ДНК- клона в Escherichia coli продуцировала функциональную и аутентичную b-N-GlcNAcase StrH, имеющую агликонспецифичность, идентичной ферменту дикого типа. Также было показано, что b-N-GlcNAcase StrH полностью гидролизует терминальные остатки GlcNAc из N-гликанов вне зависимости от того, какая ветвь в гликанах (1-3) или (1-6), (Фиг. 2.), (Samantha J.King at al., Deglycosylation of human gly coconjugates by the sequential activities of exoglycosidases expressed by Streptococcus pneumoniae, //Molecular Microbiology -2005.-V.59.-N.3. -P.961-974). In Valerie A. Clarke at al., Cloning and Expression of the b-N-Acetylglucosaminidase Gene from Streptococcus pneumoniae. Generation of truncated enzymes with modified aglycon specificity, // The Journal of Biological Chemistry - 1995.-V.270.-N.15.- P. 8805-8814), the structure of the b-N-GlcNAcase StrH gene from Streptococcus pneumoniae was first identified. The expression of an almost complete DNA clone in Escherichia coli was shown to produce a functional and authentic b-N-GlcNAcase StrH, having aglycon specificity identical to the wild-type enzyme. It was also shown that bN-GlcNAcase StrH completely hydrolyzes the terminal GlcNAc residues from N-glycans, regardless of which branch in glycans (1-3) or (1-6), (Fig. 2.), (Samantha J. King at al., Deglycosylation of human gly coconjugates by the sequential activities of exoglycosidases expressed by Streptococcus pneumoniae, // Molecular Microbiology -2005.-V.59.-N.3. -P.961-974).
Получение b-N-GlcNAcase StrH в Escherichia coli в виде
гомологичного варианта фермента ранее проводилось в исследовательских целях и раскрыто в публикации Clarke V. A. At al,// Int. J. Biochrom. -1994.-V. l .- P. 151-158. Авторы получают b-N-GlcNAcase StrH в виде фьюжен - белка с глутатион- S-трансферазой с использованием аффинной хроматографии на Glutathione Sepharose и последующим расщеплением фьюжен-белка протеазой фактором Ха. Данный метод выделения имеет свои очевидные недостатки для получения фермента в промышленных масштабах: Obtaining bN-GlcNAcase StrH in Escherichia coli as a homologous variant of the enzyme was previously conducted for research purposes and disclosed in the publication Clarke VA At al, // Int. J. Biochrom. -1994.-V. l .- P. 151-158. The authors obtain bN-GlcNAcase StrH as a fusion protein with glutathione S-transferase using affinity chromatography on Glutathione Sepharose and subsequent cleavage of the fusion protein with factor Xa protease. This extraction method has its obvious disadvantages for producing the enzyme on an industrial scale:
1. На коммерчески доступный сорбент glutathione (g- glutamylcysteinylglycine) Sepharose может повлиять g- глутамилтранспептидаза, находящаяся в неочищенных клеточных лизатах. Следовательно, глутатионный сорбент имеет конечный срок службы и может быть регенерирован и повторно использован 4-20 раз, в отличие от, например, IMAC сорбентов, которые можно использовать практически бесконечно (Michelle Е. Kimple at al., Overview of Affinity Tags for Protein Purification, HHS Public Access Author manuscript, //Curr Protoc Protein Sci.- 2015- V.73.-P. 1-26). 1. The commercially available sorbent glutathione (g-glutamylcysteinylglycine) Sepharose may be affected by g-glutamyl transpeptidase found in untreated cell lysates. Therefore, glutathione sorbent has a finite life and can be regenerated and reused 4-20 times, unlike, for example, IMAC sorbents, which can be used almost indefinitely (Michelle E. Kimple at al., Overview of Affinity Tags for Protein Purification , HHS Public Access Author manuscript, // Curr Protoc Protein Sci.- 2015- V.73.-P. 1-26).
2. Шаперонин - 70 kDa E. coli (белок теплового шока) также адсорбируется на сорбент и коэлюируется с целевым GST-фьюжен белком. 2. Chaperonin - 70 kDa E. coli (heat shock protein) is also adsorbed onto the sorbent and coelutes with the target GST fusion protein.
3. Высокий уровень экспрессии фьюжен белка GST в Е. coli может привести к накоплению агрегированного белка в телах включения. Хроматография на Glutathione Sepharose зависит от правильного рефолдинга GST. Поэтому перед очисткой должен быть проведен рефолдинг, однако большой размер GST 26 кДа и его димеризация в растворе может повлиять на свойства фьюжен белка. 3. The high level of expression of fusion GST protein in E. coli can lead to the accumulation of aggregated protein in inclusion bodies. Chromatography on a Glutathione Sepharose is dependent on proper GST refolding. Therefore, refolding must be carried out before purification, however, the large size of GST 26 kDa and its dimerization in solution can affect the properties of fusion protein.
4. Высокая стоимость фактора Ха, дополнительная очистка от фактора Ха и продуктов гидролиза, а также дополнительный контроль
наличия фактора Ха в готовом продукте значительно увеличивают себестоимость препарата. 4. The high cost of factor XA, additional purification from factor XA and hydrolysis products, as well as additional control the presence of factor Xa in the finished product significantly increases the cost of the drug.
Наиболее близким к заявленному изобретению можно считать способ очистки b-N-GlcNAcase StrH, раскрытый в работе Benjamin Pluvinage at al, Conformational Analysis of StrH, the Surface- Attached exo-b- d-N-Acetylglucosaminidase from Streptococcus pneumoniae, // Journal of Molecular Biology- 2013.- V. 425.- N. 23. -P. 334-349. Данный способ предполагает очистку гомологичного варианта фермента и его вариантов в виде фьюжен белка с полигистидиновой последовательностью, в аналитических количествах. Целевые белки очищали с помощью металлохелатной хроматографии на IMAC Sepharose с последующим гидролизом тромбином и анионообменной хроматографией на UNOsphere Q (Bio-Rad Laboratories), гельфильтрацией на Sephacryl S200(GE Biosciences) с последующим концентрированием ультрафильтрацией с использованием мембраны с отсечением 10 кДа. Данная схема очистки с использованием тромбина является неприемлемой для масштабного производства препарата в силу уже упоминаемых причин по гидролизу фактором Ха. Кроме того, авторы не сообщают об уровне содержания в препарате бактериальных эндотоксинов. Удаление бактериальных эндотоксинов является принципиальным и необходимым требованием, как с точки зрения российских, так и международных стандартов (ГФ XIII, Document Q AS/ 11.452 FINAL July 2012). Однако часто остается технически трудновыполнимой задачей, поэтому содержание бактериальных эндотоксинов в промежуточных продуктах, применяемых при производстве ЛС, является критическим параметром. При оценке качества таких промежуточных продуктов, как гликозидазы, помимо соответствия субстратной специфичности, активности, особо должны
оцениваться такие потенциально опасные контаминанты, как бактериальные эндотоксины и клеточная ДНК, в том числе потенциально онкогенная, а также гомогенность препарата. Closest to the claimed invention can be considered a method for cleaning bN-GlcNAcase StrH, disclosed in Benjamin Pluvinage at al, Conformational Analysis of StrH, the Surface-Attached exo-b-dN-Acetylglucosaminidase from Streptococcus pneumoniae, // Journal of Molecular Biology- 2013 .- V. 425.- N. 23. -P. 334-349. This method involves the purification of a homologous variant of the enzyme and its variants in the form of a fusion protein with a polyhistidine sequence, in analytical quantities. The target proteins were purified by metal chelate chromatography on an IMAC Sepharose, followed by thrombin hydrolysis and anion exchange chromatography on UNOsphere Q (Bio-Rad Laboratories), gel filtration on Sephacryl S200 (GE Biosciences), followed by concentration by ultrafiltration using a 10 kDa cut-off membrane. This purification scheme using thrombin is unacceptable for large-scale production of the drug due to the already mentioned reasons for the hydrolysis of factor Xa. In addition, the authors did not report the level of bacterial endotoxins in the preparation. The removal of bacterial endotoxins is a fundamental and necessary requirement, both from the point of view of Russian and international standards (GF XIII, Document Q AS / 11.452 FINAL July 2012). However, it often remains a technically difficult task, therefore, the content of bacterial endotoxins in the intermediate products used in the manufacture of drugs is a critical parameter. When assessing the quality of intermediates such as glycosidases, in addition to matching substrate specificity, activity, Potentially dangerous contaminants such as bacterial endotoxins and cellular DNA, including potentially oncogenic, as well as drug homogeneity, are evaluated.
Для решения задачи получения высокоочищенной b-N-GlcNAcase StrH в промышленных объемах авторы изобретения предлагают новый способ получения рекомбинантной b-N-GlcNAcase StrH из Streptococcus pneumoniae, включающий синтез оптимизированной для трансляции в Е.соЫ последовательности ДНК, кодирующий белок b-N-GlcNAcase StrH, нуклеотидная последовательность которого приведена в составе SEQ ID N01, конструирование экспрессионного плазмидного вектора, кодирующего химерный белок с N- концевой химеризацией, составленного пептидными последовательностями шести гистидинов, сайт узнавания тромбина а также 1117 аминокислотных остатков (без сигнального пептида, одного из двух G5 доменов и якорного пептида) b- N-GlcNAcase StrH из Streptococcus pneumoniae (аминокислотная последовательность представлена SEQ ID NO 2), получение рекомбинантного белка продукцией в E.coli, очистку белка из растворимой фракции клеточного лизата с помощью металлохелатной хроматографии на IMAC Sepharose, хроматографии в режиме проскока на Blue Sepharose FF, анионобменной хроматографии на Toyopearl Giga Cap Q- 65 ОМ, хроматографии в режиме проскока на Butyl Capto, концентрирующей анионобменной хроматографию на Q Sepharose FF, с выходом высокоочищенного активного препарата bϋ1oNAϋ38e StrH, пригодного для использования на производстве ЛС и гликобиологических исследований, не менее 0,04 г с литра бактериальной культуры. To solve the problem of obtaining highly purified bN-GlcNAcase StrH in industrial volumes, the inventors propose a new method for producing recombinant bN-GlcNAcase StrH from Streptococcus pneumoniae, including the synthesis of a DNA sequence optimized for translation into E. cola encoding the bN-GlcNAcase StrH protein, the nucleotide sequence of which is shown as part of SEQ ID N01, the construction of an expression plasmid vector encoding a chimeric protein with N-terminal chimerization composed of peptide sequences of six histidines, the recognition site thrombin and 1117 amino acid residues (without signal peptide, one of two G5 domains and an anchor peptide) b-N-GlcNAcase StrH from Streptococcus pneumoniae (amino acid sequence represented by SEQ ID NO 2), production of recombinant protein by products in E. coli, purification protein from the soluble fraction of the cell lysate using metal chelate chromatography on IMAC Sepharose, chromatography in slip mode on Blue Sepharose FF, anion exchange chromatography on Toyopearl Giga Cap Q-65 OM, chromatography in slip mode on Butyl Capto, concentrating anion exchange chromatograph iju on Q Sepharose FF, in a yield of highly active drug bϋ1oNAϋ38e StrH, suitable for use in the production of drugs and glikobiologicheskih research, not less than 0.04 g per liter of bacterial culture.
Для получения наиболее хроматографически гомогенного препарата (более 98 % по результатам гель-фильтрационной ВЭЖХ),
применяемого для точных гликобиологических исследований, в схему может быть дополнительно включено концентрирование ультрафильтрацией с использованием мембраны с отсечением 50 кДа и гель - фильтрация на Superdex 200. To obtain the most chromatographically homogeneous preparation (more than 98% according to the results of gel filtration HPLC), used for precise glycobiological studies, concentration by ultrafiltration using a membrane with a cut-off of 50 kDa and gel filtration on Superdex 200 can be additionally included in the scheme.
Основным техническим результатом изобретения является получение в промышленных масштабах высокоочищенного активного препарата b-N-GlcNAcase StrH, пригодного для использования на производстве ЛС и гликобиологических исследований. The main technical result of the invention is the production on an industrial scale highly purified active drug b-N-GlcNAcase StrH, suitable for use in the manufacture of drugs and glycobiological studies.
Для осуществления изобретения был создан штамм генно- инженерно-модифицированного микроорганизма E.coli ВКПМ В- 12747: штамм получен трансформацией реципиентного штамма-хозяина BL21(DE3) плазмидами pLysS, несущей ген Т7 лизоцима, являющегося ингибитором Т7 РНК полимеразы, рМТ1618 (pET15b-STRH_STRPN), несущей ген белка b-N-GlcNAcase StrH, составной частью которого являются последовательности 20 аминокислотных остатков, содержащих гексагистидиновый тэг, сайт узнавания тромбина и 1117 аминокислотных остатков (без сигнального пептида, одного из двух G5 доменов и якорного пептида) b-N-GlcNAcase StrH (Фиг.З.). To carry out the invention, a strain of a genetically modified microorganism E.coli VKPM B-12747 was created: the strain was obtained by transforming the recipient host strain BL21 (DE3) with plasmids pLysS carrying the T7 lysozyme gene, which is an inhibitor of T7 RNA polymerase, pMT1618 (pET15b-STRH_STH_STH_ ), which carries the bN-GlcNAcase StrH protein gene, of which a sequence of 20 amino acid residues containing a hexahistidine tag, a thrombin recognition site and 1117 amino acid residues (without a signal peptide, one of two G5 domains and anchor peptide tida) b-N-GlcNAcase StrH (Fig.Z.).
Рефолдинг продукта не требуется, так как он синтезируется в бактериальной цитоплазме преимущественно в растворенном состоянии при культивировании E.coli ВКПМ В- 12747 с уровнем биосинтеза вариантов целевого белка не менее 40 мг/л культуральной жидкости. Refolding of the product is not required, since it is synthesized in the bacterial cytoplasm mainly in the dissolved state during the cultivation of E. coli VKPM B-12747 with the level of biosynthesis of variants of the target protein of at least 40 mg / l of culture fluid.
Высокоэффективное извлечение целевого белка b-N-GlcNAcase StrH из клеток достигается в результате процедуры лизиса бактериальных клеток с использованием гомогенизатора высокого давления. Highly efficient extraction of the target b-N-GlcNAcase StrH protein from cells is achieved as a result of bacterial cell lysis using a high pressure homogenizer.
Получение активного, хроматографически гомогенного с низким содержанием бактериальных эндотоксинов и ДНК хозяина препарата b-
N-GlcNAcase StrH достигается благодаря схеме хроматографической очистки, включающей в себя: металлохелатную хроматографию на IMAC Sepharose 6 F F, хроматографию в режиме проскока на Blue Sepharose FF, анионобменную хроматографию на Toyopearl Giga Cap Q- 65 ОМ, хроматографию в режиме проскока на Butyl Capto, концентрирующую анионобменную хроматографию на Q Sepharose FF. Для получения наиболее хроматографически гомогенного препарата в схему может быть дополнительно включено концентрирование ультрафильтрацией с использованием мембраны с отсечением 50 кДа и гель - фильтрация на Superdex 200. Obtaining active, chromatographically homogeneous with a low content of bacterial endotoxins and host DNA of the drug b- N-GlcNAcase StrH is achieved thanks to the chromatographic purification scheme, which includes: metal chelate chromatography on IMAC Sepharose 6 FF, chromatography in slip mode on Blue Sepharose FF, anion exchange chromatography in Toyopearl Giga Cap Q-65 OM, chromatography in slip mode on Butyl Capto, concentration anion exchange chromatography on Q Sepharose FF. To obtain the most chromatographically homogeneous preparation, concentration by ultrafiltration using a membrane with a cut-off of 50 kDa and gel filtration on Superdex 200 can be additionally included in the scheme.
Введение в схему очистки стадии хроматографии на Blue Sepharose в режиме проскока позволяет значительно очистить целевой белок от контаминирующих примесей клеточных белков, коэлюирующихся с целевым белком на всех стадиях. The introduction to the purification scheme of the Blue Sepharose chromatography step in a slip mode allows one to significantly purify the target protein from contaminating impurities of cellular proteins coeluting with the target protein at all stages.
Применение TOYOPEARL GigaCap Q-650M, высокомолекулярного анионообменного сорбента с высоким разрешением, оптимизированным для захвата и очистки белков с большим молекулярным весом. Подобраны условия посадки и съема целевого белка, позволяющие максимально очистить целевой белок от контаминирующих примесей клеточных белков и липополисахаридов. Кроме того, способ отличается от ранее известных также применением хроматографии на Butyl Capto в режиме проскока, позволяющей удалить контаминирующие примеси гидрофобных белков и липополисахаридов. Материалы и методы Use of TOYOPEARL GigaCap Q-650M, a high-resolution, high molecular weight anion-exchange sorbent optimized for the capture and purification of high molecular weight proteins. The conditions of planting and removal of the target protein were selected, which allow maximum purification of the target protein from contaminating impurities of cellular proteins and lipopolysaccharides. In addition, the method differs from previously known also by the use of Butyl Capto chromatography in slip mode, which allows removing contaminants of hydrophobic proteins and lipopolysaccharides. Materials and methods
Для экспрессии b-N-GlcNAcase StrH получают генно-инженерные конструкции (ГИК) рМТ1618. Для получения ГИК, кодирующих b-N- GlcNAcase StrH, была разработана схема клонирования на базе вектора
pET-15b (Novagen), предполагающая получение полной версии, имеющей 6xHis-tag на N-конце. Для получения соответствующей ГИК в компании TOP Gene Technologies был заказан синтетический ген, кодирующий b-N- GlcNAcase StrH (геномная последовательность бактерии Streptococcus pneumoniae, кодирующая 1117 аминокислотных остатков (без сигнального пептида, одного из двух G5 доменов и якорного пептида) b- N-ацетилглюкозаминидазы (strH), слитая на 5’ -конце с частью векторной последовательности материнской плазмиды рЕТ15Ь(+), кодирующей 20 аминокислотных остатков, содержащих гексагистидиновый тэг), фланкированный сайтами рестрикции Ndel, BamHI, содержащий внутренние сайты рестрикции Nhel для получения «каталитического» домена и стоп-кодоны на З’-конце последовательности (TGATAA). В ТОР Gene Technologies была проведена кодон-оптимизация последовательности для экспрессии белка в E.coli, принимая во внимание требование об отсутствии в последовательности «внутренних» сайтов эндонуклеаз рестрикции Ndel, BamHI, Xhol и Nhel. Кодон- оптимизация полученной последовательности b-N-GlcNAcase StrH была проверена на сайте http://gcua.schoedl.de/sequential v2.html. Соответствие транслируемой аминокислотной последовательности заказанной подтверждено с помощью выравнивания в программе MEGA 6.0. Отсутствие нежелательных сайтов рестрикции подтверждено с помощью программного обеспечения CloneManager 9.0. Для получения ГИК рМТ1618 (pET15b-STRH_STRPN) была проведена наработка и рестрикционное картирование синтетического гена pAPGl lO- b-N- GlcNAcase StrH. Согласно разработанной схеме было проведено клонирование фрагмента, кодирующего стрептококковую b-N-GlcNAcase StrH из плазмиды pAPGHO-STRH STRPN, в векторную плазмиду рЕТ15Ь по сайтам рестрикции Ndel, BamHI. Полученные в ходе
рестрикции фрагменты очищали и использовали для лигирования. Лигазной смесью трансформировали химически компетентные клетки E.coli XL10-GOLD. Клетки высевали для получения трансформированных клонов. For the expression of bN-GlcNAcase StrH, genetic engineering constructs (HECs) of pMT1618 are obtained. To obtain GICs encoding bN-GlcNAcase StrH, a vector-based cloning scheme was developed pET-15b (Novagen), which assumes the receipt of the full version having 6xHis-tag at the N-terminus. To obtain the corresponding GIC, TOP Gene Technologies ordered a synthetic gene encoding bN-GlcNAcase StrH (the genomic sequence of the bacterium Streptococcus pneumoniae encoding 1117 amino acid residues (without signal peptide, one of two G5 domains and anchor peptide) b-N-acetylglucosaminidase ( strH), fused at the 5 'end with part of the vector sequence of the maternal plasmid pET15b (+) encoding 20 amino acid residues containing hexahistidine tag) flanked by Ndel restriction sites, BamHI, containing internal restriction sites and Nhel to obtain a "catalytic" domain and stop codons at the 3'-end of the sequence (TGATAA). At TOR Gene Technologies, codon optimization was performed for protein expression in E. coli, taking into account the requirement that there are no Ndel, BamHI, Xhol, and Nhel restriction endonuclease sites in the sequence. Codon optimization of the obtained bN-GlcNAcase StrH sequence was verified at http://gcua.schoedl.de/sequential v2.html. The correspondence of the translated amino acid sequence to the ordered one was confirmed by alignment in the MEGA 6.0 program. The absence of unwanted restriction sites was confirmed using CloneManager 9.0 software. To obtain the GIC pMT1618 (pET15b-STRH_STRPN), the synthesis and restriction mapping of the synthetic gene pAPGl lO-bN-GlcNAcase StrH was performed. According to the developed scheme, the fragment encoding streptococcal bN-GlcNAcase StrH from plasmid pAPGHO-STRH STRPN was cloned into the vector plasmid pET15b at the Ndel, BamHI restriction sites. Received during restriction fragments were purified and used for ligation. Chemically competent E. coli XL10-GOLD cells were transformed with a ligase mixture. Cells were seeded to obtain transformed clones.
Затем выделяли плазмидную ДНК и проводили рестрикционное картирование с использованием эндонуклеазы рестрикции Pstl. Образец плазмидной ДНК передавали на секвенирование в "Евроген". Полученная последовательность была выровнена с «теоретической» последовательностью рМТ 1618. Plasmid DNA was then isolated and restriction mapping using Pstl restriction endonuclease was performed. A plasmid DNA sample was transferred for sequencing to Eurogen. The resulting sequence was aligned with the "theoretical" sequence pMT 1618.
Для проведения аналитической экспрессии плазмидную ДНК ГИК рМТ1618 трансформировали в штаммы BL21(DE3) pLysS. На основании аналитической экспрессии отбирали клоны-суперпродуценты b-N- GlcNAcase StrH. Затем получали штамм-суперпродуцент b-N-GlcNAcase StrH. Штамм был депонирован во Всероссийской коллекции промышленных микроорганизмов (ВКПМ) под инвентарным номером - ВКПМ В- 12747. For analytical expression, plasmid DNA GIC pMT1618 was transformed into strains BL21 (DE3) pLysS. The super-producing clones of b-N-GlcNAcase StrH were selected based on analytical expression. Then received the super-producer strain b-N-GlcNAcase StrH. The strain was deposited in the All-Russian collection of industrial microorganisms (VKPM) under inventory number - VKPM B-12747.
Для получения целевого белка b-N-GlcNAcase StrH проводили препаративную экспрессию путем культивирования E.coli ВКПМ В- 12747 в полноценной питательной среде с относительным содержанием растворенного кислорода не менее 10 % при температуре 37°С, значении pH - 6, 5-7, 6. Длительность процесса культивирования составляла 5-6 часов. Благодаря обогащенной культуральной среде, в течение первого этапа происходил активный рост клеток. Присутствие в среде глюкозы подавляет экспрессию рекомбинантного белка, а, следовательно, снижает нагрузку на клетку штамма-продуцента, что приводит к увеличению количества накапливаемой биомассы. To obtain the target protein bN-GlcNAcase StrH, preparative expression was carried out by culturing E. coli VKPM B-12747 in a complete nutrient medium with a relative dissolved oxygen content of at least 10% at a temperature of 37 ° C and a pH value of 6, 5-7, 6. The duration of the cultivation process was 5-6 hours. Due to the enriched culture medium, during the first stage, active cell growth occurred. The presence of glucose in the medium inhibits the expression of the recombinant protein, and, therefore, reduces the load on the cell of the producer strain, which leads to an increase in the amount of accumulated biomass.
С 2,5-3 часа культивирования начинается этап биосинтеза гибридного белка. Экспрессия белка b-N-GlcNAcase StrH находится под
контролем промотора, который дерепрессируется внесением раствора ИПТГ до конечной концентрации 0,0476 мас.%. Уровень биосинтеза вариантов целевого белка b-N-GlcNAcase StrH составляет не менее 15 мг/л культуральной жидкости. From 2.5-3 hours of cultivation, the stage of biosynthesis of the hybrid protein begins. The expression of bN-GlcNAcase StrH protein is under the control of the promoter, which is depressed by introducing a solution of IPTG to a final concentration of 0.0476 wt.%. The level of biosynthesis of the variants of the target bN-GlcNAcase StrH protein is at least 15 mg / l of culture fluid.
Бактериальную биомассу собирали центрифугированием и хранили при - 70°С. Клеточную биомассу лизировали, лизат осветляли центрифугированием и проводили выделение белка b-N-GlcNAcase StrH из лизата путем связывания целевого белка металлохелатной хроматографией на колонке с
IMAC Sepharose FF. Доочищали фермент последовательными хроматографиями на Blue Sepharose FF в режиме проскока, анионообменной Toyopearl Giga Cap Q- 650M, Butyl Capto в режиме проскока, хроматографией на Q HP, концентрированием и гельфильтрацией. На каждом этапе контроль степени очистки проводят электрофорезом в ПААТ. Bacterial biomass was collected by centrifugation and stored at - 70 ° C. Cellular biomass was lysed, the lysate was clarified by centrifugation, and bN-GlcNAcase StrH protein was isolated from the lysate by binding to the target protein by column chelate chromatography IMAC Sepharose FF. The enzyme was further purified by sequential chromatography on Blue Sepharose FF in slip mode, anion exchange Toyopearl Giga Cap Q-650M, Butyl Capto in slip mode, chromatography on Q HP, concentration and gel filtration. At each stage, the degree of purification is monitored by electrophoresis in PAAT.
Выход белка составляет не менее 0,04 мг на литр бактериальной культуры. The protein yield is at least 0.04 mg per liter of bacterial culture.
Конечный продукт, полученный способ по изобретению, имеет следующие параметры: The final product, the obtained method according to the invention, has the following parameters:
- содержание белка (УФ-спектрофотометрия) - не менее 0,5 мг/мл; - содержание бактериальных эндотоксинов (ГФ XIII, гель-тромб тест с ЛАЛ-реактивомОФС.1.2.4.0006.15) не более 100 ЭЕ/мг; - protein content (UV spectrophotometry) - not less than 0.5 mg / ml; - the content of bacterial endotoxins (HF XIII, gel thrombus test with LAL reagent OFS.1.2.4.0006.15) not more than 100 IU / mg;
- Хроматографическая чистота (ГФ ВЭЖХ) не менее 85 %; - Chromatographic purity (HPLC) not less than 85%;
- Ферментативная активность (Колориметрический метод, b-N- Acetylglucosaminidase Assay Kit” (Sigma)) не менее 150 U/мг; - Enzymatic activity (Colorimetric method, b-N-Acetylglucosaminidase Assay Kit ”(Sigma)) at least 150 U / mg;
- Остаточная ДНК штамма-продуцента (Количественный ПЦР в реальном времени) не более 20 пг/мг; - Residual DNA of the producer strain (Real-time quantitative PCR) not more than 20 pg / mg;
С использованием методов очистки настоящего изобретения удалось получить препарат b-N-GlcNAcase StrH с хроматографической
чистотой, определенной с помощью гель-фильтрационной ВЭЖХ, 90% гомогенности и более высокоочищенный - 98,8%-ной гомогенности. Using the purification methods of the present invention, a chromatographic bN-GlcNAcase StrH preparation was obtained. purity determined by gel filtration HPLC, 90% homogeneity and more highly purified - 98.8% homogeneity.
Удельная активность препаратов b-N-GlcNAcase StrH по настоящему изобретению составляет от 230 единиц/мг белка и выше. The specific activity of the b-N-GlcNAcase StrH preparations of the present invention is from 230 units / mg protein and above.
Содержание бактериальных эндотоксинов в препарате b-N- The content of bacterial endotoxins in the preparation b-N-
GlcNAcase StrH по настоящему изобретению составляет не более 4 ЕЭ /мг белка. The GlcNAcase StrH of the present invention is not more than 4 UE / mg protein.
Содержание остаточной ДНК штамма продуцента в препарате b-N- GlcNAcase StrH настоящего изобретения предпочтительно составляет не более 4,0 пг/мг белка. The residual DNA of the producer strain in the b-N-GlcNAcase StrH preparation of the present invention is preferably not more than 4.0 pg / mg protein.
Получение b-N-GlcNAcase StrH на основе оптимизированной синтетической последовательности ДНК с короткой последовательностью из шести гистидинов на N- конце рекомбинантного белка происходит без удаления гексагистидинового тэга, что позволяет упростить очистку и удалить из схемы очистки применение дорогостоящих препаратов для удаления тэга. Аминокислотная последовательность b-N-GlcNAcase StrH представлена SEQ ID NO 2. В качестве первой стадии авторы изобретения применяют металлохелатную хроматографию на IMAC Sepharose 6 FF для полной сорбции целевого белка с гексагистидиновым тэгом. Применение на этой стадии промывок кислым буфером с высокой ионной силой позволяет в значительной степени удалить примеси клеточных белков из целевого продукта. Применение хроматографии на Blue Sepharose в режиме проскока позволяет очистить целевой белок от контаминирующих примесей клеточных белков, коэлюирующихся с целевым белком на всех стадиях. Анионообменную хроматографию проводят на Toyopearl Giga Cap Q-650M, высокомолекулярном анионообменном сорбенте с высоким разрешением, оптимизированным для захвата и очистки белков с
большим молекулярным весом. Условия посадки и съема целевого белка подобраны таким образом, чтобы максимально очистить целевой белок от контаминирующих примесей клеточных белков и бактериальных эндотоксинов. Хроматография в режиме проскока на Butyl Capto позволяет удалить контаминирующие примеси гидрофобных белков и бактериальных эндотоксинов. Финальная концентрирующая анионообменная хроматография на Q Sepharose FF позволяет получить высокоочищенный активный препарат b-N-GlcNAcase StrH, пригодный для использования на производстве ЛС и гликобиологических исследований, с выходом не менее 0,04 г с литра бактериальной культуры. The preparation of bN-GlcNAcase StrH based on an optimized synthetic DNA sequence with a short sequence of six histidines at the N-terminus of the recombinant protein occurs without removing the hexahistidine tag, which simplifies the purification and removes the use of expensive tag removers from the purification scheme. The amino acid sequence of bN-GlcNAcase StrH is represented by SEQ ID NO 2. As a first step, the inventors use IMAC Sepharose 6 FF metal chelate chromatography to completely sorb the target protein with a hexahistidine tag. The use at this stage of washing with acidic buffer with high ionic strength can significantly remove impurities of cellular proteins from the target product. The use of chromatography on Blue Sepharose in slip mode allows you to clean the target protein from contaminating impurities of cellular proteins coeluting with the target protein at all stages. Anion-exchange chromatography is carried out on Toyopearl Giga Cap Q-650M, a high-resolution, high-molecular-weight anion-exchange sorbent optimized for the capture and purification of proteins with high molecular weight. The conditions for planting and removing the target protein are selected in such a way as to maximize purification of the target protein from contaminating impurities of cellular proteins and bacterial endotoxins. Breakthrough chromatography on Butyl Capto removes contaminating contaminants of hydrophobic proteins and bacterial endotoxins. Final concentration anion exchange chromatography on Q Sepharose FF allows to obtain a highly purified active preparation bN-GlcNAcase StrH, suitable for use in the manufacture of drugs and glycobiological studies, with a yield of at least 0.04 g per liter of bacterial culture.
С использованием методов очистки настоящего изобретения удалось получить препарат b-N-GlcNAcase StrH с хроматографической чистотой, определенной с помощью гель-фильтрационной ВЭЖХ 90,0 %- ной гомогенности (Фиг.7.). Using the purification methods of the present invention, it was possible to obtain a b-N-GlcNAcase StrH preparation with chromatographic purity determined by gel filtration HPLC of 90.0% homogeneity (Fig. 7).
Для получения наиболее хроматографически гомогенного препарата (более 98 % по результатам гель-фильтрационной ВЭЖХ, Фиг.8), применяемого для точных гликобиологических исследований, в схему может быть дополнительно включено концентрирование ультрафильтрацией с использованием мембраны с отсечением 50 кДа и гель - фильтрация на Superdex 200. To obtain the most chromatographically homogeneous preparation (more than 98% according to the results of gel filtration HPLC, Fig. 8), used for accurate glycobiological studies, concentration by ultrafiltration using a membrane with a cutoff of 50 kDa and gel filtration on Superdex 200 can be additionally included in the scheme .
Удельная активность препаратов b-N-GlcNAcase StrH, полученных раскрытым в изобретении способом, составляет 240 единиц/мг белка; содержание бактериальных эндотоксинов составляет от 2 ЕЭ /мг до 4 ЕЭ /мг белка; содержание остаточной ДНК штамма продуцента составляет не более 4,0 пг/мг белка. The specific activity of b-N-GlcNAcase StrH preparations obtained by the method disclosed in the invention is 240 units / mg protein; the content of bacterial endotoxins is from 2 U / mg to 4 U / mg protein; the residual DNA content of the producer strain is not more than 4.0 pg / mg protein.
Заявленный способ позволяет получить препарат рекомбинантной b-N-GlcNAcase StrH, имеющий более высокую степень чистоты по сравнению с коммерческими аналогами, результаты сравнения
представлены в Таблице 2. The claimed method allows to obtain a preparation of recombinant bN-GlcNAcase StrH, having a higher degree of purity compared with commercial analogues, the comparison results are presented in Table 2.
Таблица 2. Сравнение качества полученных препаратов рекомбинантной b-N-GlcNAcase StrH и коммерческого препарата b-N- GlcNAcase Sigma Aldrich Table 2. Comparison of the quality of the obtained preparations of recombinant b-N-GlcNAcase StrH and commercial preparation b-N-GlcNAcase Sigma Aldrich
Для лучшего понимания сущности изобретения ниже следуют примеры его конкретного исполнения: For a better understanding of the invention, the following are examples of its specific implementation:
Пример 1. Микробиологический синтез белка b-N-GkNAcase StrH Example 1. Microbiological synthesis of protein b-N-GkNAcase StrH
Заявленный способ реализован для штамма-продуцента E.coli ВКПМ В- 12747 при культивировании в ферментере (Biotron LiFlus SP/SL-20L, Юж. Корея) в объеме питательной среды 15 л. pH поддерживают на уровне 7, 0-7, 6. Культивирование ведут при температуре 37 °С. Относительное содержание растворенного кислорода поддерживают на уровне 20-100 % путем перемешивания со скоростью
800 об/мин и подачи воздуха с объемным расходом 15 л/мин. Инокулят в объеме 0,75 л, выращенный до оптической плотности, измеренной при длине волны 600 нм (ОД600), равной 1,5 о.е. (оптических единиц), вносят в ферментер со стерильной питательной средой. The claimed method is implemented for the producer strain E. coli VKPM B-12747 when cultured in a fermenter (Biotron LiFlus SP / SL-20L, South Korea) in a volume of nutrient medium of 15 l. pH is maintained at a level of 7, 0-7, 6. Cultivation is carried out at a temperature of 37 ° C. The relative content of dissolved oxygen is maintained at a level of 20-100% by mixing at a speed 800 rpm and air supply with a volume flow of 15 l / min. Inoculum in a volume of 0.75 l, grown to an optical density measured at a wavelength of 600 nm (OD600) equal to 1.5 p.u. (optical units), contribute to the fermenter with a sterile culture medium.
В ходе процесса культивирования производят мониторинг основных параметров роста культуры: ОД600, pH, морфология клеток, относительное содержание растворенного кислорода. На 2,5 час культивирования, когда оптическая плотность культуры достигает 3,2 о.е., вносят индуктор - стерильный раствор изопропил-b-O- 1 - тиогалактопиранозида до конечной концентрации 0,0476 мас.% (2 мМ). Культивирование проводят на протяжении 5-6 часов. Конечная оптическая плотность -28,2 о.е., выход биомассы - 44 г/л. During the cultivation process, the main parameters of culture growth are monitored: OD600, pH, cell morphology, relative dissolved oxygen content. For 2.5 hours of cultivation, when the optical density of the culture reaches 3.2 p.u., an inducer is introduced - a sterile solution of isopropyl-b-O-1 - thiogalactopyranoside to a final concentration of 0.0476 wt.% (2 mm). Cultivation is carried out for 5-6 hours. The final optical density is 28.2 pu, the biomass yield is 44 g / l.
Продуктивность по целевому продукту штамма E.coli ВКПМ В- 12747 составляет 40 мг/л культуральной жидкости. На Фиг.4. представлена динамика накопления биомассы при культивировании штамма- продуцента E.coli ВКПМ В- 12747. The productivity of the target product of E. coli strain VKPM B-12747 is 40 mg / l of culture fluid. Figure 4. The dynamics of biomass accumulation during cultivation of the producer strain E. coli VKPM B-12747 is presented.
Пример 2. Выделение и очистка белка b-N-GlcNAcase StrH из бактериальной биомассы. Получение промышленной серии препарата b-N-GlcNAcase StrH. Example 2. Isolation and purification of b-N-GlcNAcase StrH protein from bacterial biomass. Obtaining an industrial series of the drug b-N-GlcNAcase StrH.
2.1 Дезинтеграция биомассы. 2.1 Disintegration of biomass.
Буфер А - 20 mM Tris-HCl, 0.5 М NaCl, pH 7.8; Buffer A - 20 mM Tris-HCl, 0.5 M NaCl, pH 7.8;
Замороженную биомассу клеток 180 г, ЗХР по 60 г суспендируют в 350 мл буфера А с PMSF до 0,003% в течение 45 мин на льду и разрушают в проточном гомогенизаторе высокого давления Panda Plus 2000(GEA Niro Soavi). Гомогенизатор промывают 250 мл охлажденной до 0 °С водой очищенной, настраивают по охлажденному до 0 °С буферу А, настройка II ступени - 140 бар, настройка I ступени - 800 бар, затем
лизируют суспензию клеток в этом режиме, собирая лизат в емкость, помещенную в ледяную баню, промывают гомогенизатор 30 мл буфера А. Лизис повторяют еще 2 раза в том же режиме. Лизат объемом 1500 мл осветляют центрифугированием 33250 х g (JLA-16.250) при 4°С в течение 60 мин. Frozen cell biomass of 180 g, 60 g of ZXR were suspended in 350 ml of buffer A with PMSF up to 0.003% for 45 min on ice and destroyed in a Panda Plus 2000 high-pressure flow homogenizer (GEA Niro Soavi). The homogenizer is washed with 250 ml of purified water cooled to 0 ° C, adjusted to buffer A cooled to 0 ° C, setting of stage II is 140 bar, setting of stage I is 800 bar, then lyse the cell suspension in this mode, collecting the lysate in a container placed in an ice bath, rinse the homogenizer with 30 ml of buffer A. The lysis is repeated 2 more times in the same mode. A 1500 ml lysate is clarified by centrifugation at 33250 x g (JLA-16.250) at 4 ° C for 60 minutes.
Хроматографии проводят при 4°С, хроматографическая система Akta Purifer 100 (GE). Chromatography is carried out at 4 ° C, Akta Purifer 100 (GE) chromatographic system.
2.2. Хроматография на Ni2+ IMAC Sepharose FF. 2.2. Chromatography on Ni 2+ IMAC Sepharose FF.
Супернатант 1500 мл, разбавляют буфером А до 1800мл, доводят pH до 7,8 2М NaOH, фильтруют через глубинный фильтр Sartopor 2 150 и наносят на колонку с Ni IMAC Sepharose, уравновешенную буфером А. После нанесения колонку промывают буфером А и натрий -ацетатным буфером с 1 М NaCl, pH 5.0. Целевой белок элюируют 250 мМ имидазола. The supernatant is 1500 ml, diluted with buffer A to 1800 ml, adjusted to pH 7.8 with 2M NaOH, filtered through a Sartopor 2 150 depth filter and applied to a Ni IMAC Sepharose column equilibrated with buffer A. After application, the column is washed with buffer A and sodium acetate buffer with 1 M NaCl, pH 5.0. The target protein is eluted with 250 mM imidazole.
2.3 Хроматография на Blue Sepharose 6 FF 30 мл в режиме проскока. Элюат с Ni IMAC Sepharose разбавляют стартовым буфером (50 mM MES, pH 6,5) до проводимости 5,7 mSm/sm, доводят pH до 6,5, и наносят на колонку с Blue Sepharose 6 FF 30 мл, уравновешенную стартовым буфером. Целевой белок собирают в режиме проскока. 2.3 Chromatography on Blue Sepharose 6 FF 30 ml in slip mode. Sepharose Ni IMAC eluate was diluted with start buffer (50 mM MES, pH 6.5) to a conductivity of 5.7 mSm / sm, adjusted to pH 6.5, and applied to a 30 ml Blue Sepharose 6 FF column equilibrated with start buffer. The target protein is collected in slip mode.
2.4 Хроматография на Toyopearl Giga Cap Q- 65 ОМ. 2.4 Chromatography on Toyopearl Giga Cap Q-65 OM.
Проскок с Blue Sepharose 6 FF разбавляют охлажденной до 4°С водой очищенной до проводимости 1,8 mSm/sm, доводят pH до 6,5 и наносят на колонку с Toyopearl Giga Cap Q- 650M уравновешенную буфером 50 mM MES, pH 6,5. Целевой белок элюируют градиентом от ЗмМ до 0,3 М NaCl в стартовом буфере. The breakthrough with Blue Sepharose 6 FF is diluted with purified water to 4 ° C purified to a conductivity of 1.8 mSm / sm, adjusted to pH 6.5 and applied to a Toyopearl Giga Cap Q-650M column equilibrated with 50 mM MES buffer, pH 6.5 . The target protein is eluted with a gradient from 3MM to 0.3 M NaCl in the start buffer.
2.5 Концентрирование на Q Sepharose FF с предколонкой HiTrap 2.5 Concentrating on Q Sepharose FF with HiTrap Pre-Column
Butyl Capto 5 мл. Butyl Capto 5 ml.
Элюат с Toyopearl Giga Cap Q- 65 ОМ разбавляют водой очищенной до проводимости 2,04 mSm/sm, доводят pH до 7,5 и наносят на колонки с
Q Sepharose FF с предколонкой HiTrap Butyl Capto, уравновешенные буфером 20 mM Tris -НО, pH 7.5. Отмывают стартовым буфером до выхода элюата не сорбировавшихся компонентов, колонку с HiTrap Butyl Capto отсоединяют, отмывают буфером В (20 mM Tris -НС1, ImM EDTA, pH 7.5) и элюируют 250 mM NaCl в буфере В. The eluate with Toyopearl Giga Cap Q-65 OM is diluted with purified water to a conductivity of 2.04 mSm / sm, adjusted to pH 7.5 and applied to columns with Q Sepharose FF with HiTrap Butyl Capto pre-column, equilibrated with 20 mM Tris-HO buffer, pH 7.5. Washed with starting buffer until the adsorbed components were eluated, the HiTrap Butyl Capto column was disconnected, washed with buffer B (20 mM Tris-HCl, ImM EDTA, pH 7.5) and eluted with 250 mM NaCl in buffer B.
2.6 Разведение, стерилизующая фильтрация и фасовка. 2.6 Dilution, sterilizing filtration and packaging.
Элюат с Q Sepharose FF разбавляют буфером В до получения готовой формы и стерильно фильтруют через FILTER-MAX,“rapid” - 150 ml, 0.22 pm, аликвотируют и хранят при минус 20° С. The eluate with Q Sepharose FF was diluted with buffer B to the finished form and sterilely filtered through FILTER-MAX, “rapid” - 150 ml, 0.22 pm, aliquoted and stored at minus 20 ° C.
Получено: Received:
73.6 мг, 118,0 мл, 280 = 1 ,07, С- 0.624 мг/мл, 73.6 mg, 118.0 ml, 280 = 1, 07, C - 0.624 mg / ml,
Содержание бактериальных эндотоксинов: От 9,6 до 19,2 ЕЭ/мл. The content of bacterial endotoxins: From 9.6 to 19.2 EU / ml.
Активность на синтетическом субстрате: 234 U/мг. Activity on a synthetic substrate: 234 U / mg.
Чистота образца по ГФ ВЭЖХ составляет: 90,0%. The purity of the sample by GF HPLC is: 90.0%.
Остаточная ДНК штамма продуцента (E.colli): < 4,0 пг/мг. Residual DNA of producer strain (E. colli): <4.0 pg / mg.
Пример 3. Получение белка b-N-GlcNAcase StrH с улучшенными качественными характеристиками. Example 3. Obtaining protein b-N-GlcNAcase StrH with improved quality characteristics.
Гель- фильтрацию проводят при RT, хроматографическая система Akta Pure, GE. Gel filtration is carried out at RT, Akta Pure chromatography system, GE.
Фракции с концентрирующей анионообменной хроматографии, содержащие целевой белок (получение по примеру 2) объединяют и концентрируют на центриконах Amicon Ultrac, 50 К. Затем концентрат наносят на колонку с Superdex 200, используя в качестве мобильной фазы буфер 20 mM Tris НС1, 0.05 М NaCl, ImM EDTA pH 7.5. Элюцию целевого белка проводят в том же буфере. Профиль элюциии представлен на Фиг.5. Затем элюированный целевой белок b-N-GlcNAcase StrH разбавляют буфером подвижной фазы до концентрации белка не более 1
мг/мл и, соблюдая асептические условия, стерильно фильтруют через фильтр 0,22 мкм, фасуют на аликвоты и хранят при - 20° С. Concentrated anion exchange chromatography fractions containing the target protein (prepared according to Example 2) were combined and concentrated on Amicon Ultrac 50 Centricons. Then, the concentrate was applied to a Superdex 200 column using 20 mM Tris HCl, 0.05 M NaCl buffer as a mobile phase, ImM EDTA pH 7.5. The elution of the target protein is carried out in the same buffer. The elution profile is shown in FIG. 5. Then the eluted target bN-GlcNAcase StrH protein is diluted with the mobile phase buffer to a protein concentration of not more than 1 mg / ml and, observing aseptic conditions, sterilely filtered through a 0.22 μm filter, Packed in aliquots and stored at - 20 ° C.
Пример 4. Анализ активности рекомбинантной b-N-GlcNAcase StrH. Example 4. Analysis of the activity of recombinant b-N-GlcNAcase StrH.
Определение активности рекомбинантной b-N-GlcNAcase StrH проводят постановкой теста на синтетическом субстрате NAG (4- Nitrophenyl N-acetyl-P-D-glucosaminide) используя набор “b-N- Acetylglucosaminidase Assay Kit” (Sigma). В результате гидролиза субстрата NAG (4-Nitrophenyl N-acetyl^-D-glucosaminide) ферментом b- N-Acetylglucosaminidase образуется продукт p-nitrophenol, содержание которого измеряется колориметрически при длине волны 405 нм. Одна единица активности (1 unit) должна обеспечить гидролиз 1.0 pmole субстрата 4-Nitrophenyl N-acetyl^-D-glucosaminide с образованием продуктов p-nitrophenol и N-acetyl^-D-glucosaminide за 1 минуту при pH 4.7 и температуре 37°С. Для проведения теста готовят раствор субстрата «4-Nitrophenyl N-acetyl^-D-glucosaminide» с концентрацией 1 мг/мл. Для этого растворяют 10 мг субстрата в 10 мл раствора 0.09М «Citrate Buffer Solution». В качестве положительного контроля готовят раствор «b-N- Acetylglucosaminidase from Jack beans» путем разведения в 100 раз в растворе «Dilution Buffer». Готовят раствор «Stop Solution» путем разведения содержимого флакона в 118мл воды очищенной. В качестве контроля готовят разведения b-N- Acetylglucosaminidase (NAG) (Sigma, cat#A6805) в 50, 100, 200, 400 раз в растворе «Dilution Buffer». Determination of the activity of the recombinant b-N-GlcNAcase StrH is carried out by staging a test on a synthetic NAG substrate (4-Nitrophenyl N-acetyl-P-D-glucosaminide) using the “b-N-Acetylglucosaminidase Assay Kit” (Sigma). Hydrolysis of the NAG substrate (4-Nitrophenyl N-acetyl ^ -D-glucosaminide) by the b-N-Acetylglucosaminidase enzyme produces the product p-nitrophenol, the content of which is measured colorimetrically at a wavelength of 405 nm. One unit of activity (1 unit) should provide hydrolysis of 1.0 pmole of 4-Nitrophenyl N-acetyl ^ -D-glucosaminide substrate to produce p-nitrophenol and N-acetyl ^ -D-glucosaminide products in 1 minute at pH 4.7 and a temperature of 37 ° C . For the test, prepare a solution of the substrate "4-Nitrophenyl N-acetyl ^ -D-glucosaminide" with a concentration of 1 mg / ml To do this, dissolve 10 mg of the substrate in 10 ml of a 0.09M Citrate Buffer Solution solution. As a positive control, a b-N-Acetylglucosaminidase from Jack beans solution is prepared by diluting 100 times in a Dilution Buffer solution. Prepare a Stop Solution by diluting the contents of the vial in 118 ml of purified water. As a control, dilutions of b-N-Acetylglucosaminidase (NAG) (Sigma, cat # A6805) were prepared 50, 100, 200, 400 times in Dilution Buffer.
Готовят разведения очищенной рекомбинантной b-N-GlcNAcase Prepared dilutions of purified recombinant b-N-GlcNAcase
StrH в 50, 100, 200, 400 раз в растворе «Dilution Buffer». Перед внесением в лунки 96-луночного планшета 5 мкл раствора «Standard Solution» (10mM p-Nitrophenol) разбавляют в 995 мкл раствора «Stop Solution».
В лунки планшета вносят 98 мкл раствора субстрата, вносят по 2 мкл исследуемых образцов, раствора NAG и положительного контроля. Разбавленный стандартный раствор вносят по 300 мкл в лунку. В качестве «Blank» используют лунки, в которые внесено по 100 мкл раствора субстрата. Схема проведения реакции представлена в Таблице 3. StrH 50, 100, 200, 400 times in the Dilution Buffer solution. Before adding to the wells of a 96-well plate, 5 μl of Standard Solution (10mM p-Nitrophenol) was diluted in 995 μl of Stop Solution. 98 μl of the substrate solution is added to the wells of the tablet, 2 μl of the test samples, NAG solution and positive control are added. Diluted standard solution is added 300 μl per well. As "Blank" use wells, in which 100 μl of substrate solution is added. The reaction scheme is shown in Table 3.
Таблица 3. Схема проведения ферментативной реакции. Table 3. Scheme of the enzymatic reaction.
Инкубируют планшет при температуре 37°С в течение 11 минут. Вносят по 200 мкл раствора «Stop Solution» в лунки, кроме лунок со стандартным раствором. Incubate the plate at a temperature of 37 ° C for 11 minutes. Pipette 200 μl of Stop Solution into the wells, except for wells with a standard solution.
Измеряют оптическую плотность растворов при длине волны 405 нм. The optical density of the solutions is measured at a wavelength of 405 nm.
Активность фермента вычисляют по формуле: The enzyme activity is calculated by the formula:
Units/ml= (Asample-Ablank)*0,05*0,3*DF/Astandard/time/V, где Units / ml = (Asample-Ablank) * 0.05 * 0.3 * DF / Astandard / time / V, where
Asample— оптическая плотность исследуемых образцов, 405 нм Ablank - оптическая плотность раствора «Blank», 405 нм Asample — optical density of the studied samples, 405 nm; Ablank — optical density of the Blank solution, 405 nm
0.05 - pmole/ml p-Nitrophenol в стандартном растворе 0.05 - pmole / ml p-Nitrophenol in standard solution
0.3 - финальный объем реакционной смеси в лунке после добавления раствора «Stop Solution», мл 0.3 - the final volume of the reaction mixture in the well after adding the solution "Stop Solution", ml
DF - коэффициент разбавления DF - dilution ratio
A standard - оптическая плотность стандартного раствора, 405 нм time - время инкубации, мин A standard - optical density of a standard solution, 405 nm time - incubation time, min
V - объем образцов, мл
Результаты тестирования b-N-GlcNAcase StrH представлены в Таблице 4. V - volume of samples, ml The test results of bN-GlcNAcase StrH are presented in Table 4.
Таблица 4. Результаты тестирования b-N-GlcNAcase StrH на ферментативную активность Table 4. Results of testing b-N-GlcNAcase StrH for enzymatic activity
Пример 5. Электрофоретическое разделение белков Example 5. Electrophoretic separation of proteins
Электрофорез проводят в 4 - 20 % градиентном ПААГ в восстанавливающих условиях, окрашивают солями серебра (Thermo Scientific). Electrophoresis is carried out in a 4-20% gradient PAGE under reducing conditions, stained with silver salts (Thermo Scientific).
Пробоподготовку образцов проводят разведением образцов до концентрации ~50 мкг/мл добавлением буфера для нанесения проб (S.b.) с добавлением 2 М раствора дитиотреитола (ДТТ). Прогревают в течение 5 минут при (100 ± 2) ОС. Центрифугируют 1 мин., 13000 об/мин и наносят пробы (растворы, полученные при разведении образцов) по 10 мкл в лунку геля, маркеры молекулярной массы - 5 мкл в лунку геля.
Электрофорез проводят при напряжении - 400 В, силе тока - 30 мА, время - до выхода красителя. Затем окрашивают солями серебра по инструкции к набору Thermo Scientific. Результаты представлены наSample preparation of samples is carried out by diluting the samples to a concentration of ~ 50 μg / ml by adding a buffer for applying samples (Sb) with the addition of a 2 M solution of dithiothreitol (DTT). Warm up for 5 minutes at (100 ± 2) OS. Centrifuged for 1 min., 13000 rpm and apply samples (solutions obtained by diluting the samples) 10 μl per well of gel, molecular weight markers - 5 μl per well of gel. Electrophoresis is carried out at a voltage of 400 V, current strength of 30 mA, the time until the dye is released. Then stained with silver salts according to the instructions for the Thermo Scientific kit. Results are presented on
Фиг.6. 6.
Пример 6. Тестирование очищенных препаратов рекомбинантной b-N-GlcNAcase StrH в сравнении с коммерческим препаратом b-N-GlcNAcase на содержание бактериальных эндотоксинов. Example 6. Testing purified preparations of recombinant b-N-GlcNAcase StrH in comparison with the commercial preparation of b-N-GlcNAcase Case for the content of bacterial endotoxins.
Анализ на содержание бактериальных эндотоксинов проводят методом гель-тромб теста в соответствии с ГФ XIII, гель-тромб тест с ЛАЛ-реактивом ОФС.1.2.4.0006.15. Образцы разводят водой для лал- теста («Пиротест», Россия). Используют для работы: LAL-реактив Endosafe (Charles River, USA) с чувствительностью 0,03 ЕЭ/мл; Контрольный стандартный образец эндотоксина (Charles River, USA) с содержанием 20 ЕЭ/мл. В пробирки для анализа помещают по 100 мкл образцов и по 100 мкл LAL-реактива и инкубируют в течение 60 мин при температуре 37 ОС. Положительный результат: образование плотного сгустка геля. При переворачивании пробирки на 180 градусов гель не разрушается. Отрицательный результат: гель не образуется или разрушается при переворачивании пробирки. Analysis of the content of bacterial endotoxins is carried out by the method of gel-thrombus test in accordance with GF XIII, gel-thrombus test with LAL-reagent OFS.1.2.4.0006.15. Samples are diluted with water for lal test (Pirotest, Russia). Used for work: LAL-reagent Endosafe (Charles River, USA) with a sensitivity of 0.03 EU / ml; Control standard sample of endotoxin (Charles River, USA) with a content of 20 EU / ml 100 μl of samples and 100 μl of LAL reagent are placed in assay tubes and incubated for 60 min at 37 ° C. Positive result: the formation of a dense clot of gel. When the tube is turned 180 degrees, the gel does not break. Negative result: the gel does not form or breaks when the tube is turned over.
Содержание эндотоксинов в конечной точке выражается в ЕЭ/мл и рассчитывается по формуле: «С» = (разведение в конечной точке) х (чувствительность LAL-реактива). Полученные результаты представлены в Таблице 5. The endotoxin content at the endpoint is expressed in EE / ml and is calculated by the formula: “C” = (dilution at the endpoint) x (sensitivity of the LAL reagent). The results obtained are presented in Table 5.
Таблица 5. Результат гель-тромб теста на содержание бактериальных эндотоксинов. Сравнение коммерческого препарата и полученного препарата рекомбинантной b-N-GlcNAcase StrH.
Table 5. The result of a gel clot test for the content of bacterial endotoxins. Comparison of a commercial preparation and the resulting recombinant bN-GlcNAcase StrH preparation.
Примечания к таблице: Notes to the table:
«+» - положительный результат; «— » - отрицательный результат. Положительный результат: образование плотного сгустка геля. При переворачивании пробирки на 180 градусов гель не разрушается. Отрицательный результат: гель не образуется или разрушается при переворачивании пробирки. "+" - a positive result; "- " - negative result. Positive result: the formation of a dense clot of gel. When the tube is turned 180 degrees, the gel does not break. Negative result: the gel does not form or breaks when the tube is turned over.
Содержание эндотоксинов в конечной точке выражается в ЕЭ/мл и рассчитывается по формуле: «С» = (разведение в конечной точке) * (чувствительность LAL-реактива). The endotoxin content at the endpoint is expressed in EE / ml and is calculated by the formula: “C” = (dilution at the endpoint) * (sensitivity of the LAL reagent).
Пример 7. Тестирование хроматографической чистоты очищенных препаратов рекомбинантной b-N-GlcNAcase StrH в сравнении с коммерческим препаратом b-N-GIcNAcase. Example 7. Testing the chromatographic purity of purified preparations of recombinant b-N-GlcNAcase StrH in comparison with the commercial preparation b-N-GIcNAcase.
Тестирование производят методом ВЭЖХ гель- фильтрации на колонке с Superdex200 Increase 10/300 GL (GE) на хроматографической системе ВЭЖХ Alliance 2695, UV/Visible Detector 2487 (Waters). В качестве подвижной фазы используют буфер 0,02 М NaH2P04; 0,3 М
NaCl; pH = 7.0. Очищенный препарат b-N-GlcNAcase StrH вносят без разведения, коммерческий до внесения в систему разводят в 2 раза подвижной фазой. Полученные результаты анализа представлены на Фиг.7. Тестирование очищенного препарата рекомбинантной b-N- GlcNAcase StrH в сравнении с коммерческим препаратом b-N-GlcNAcase проводят таким же образом. Сравнение хроматографических профилей испытуемых образцов показывает (Фиг.8.), что b-N-GlcNAcase StrH StrH от МБЦ «Генериум» существенно более однородна по составу, тогда как коммерческая b-N-GlcNAcase более гетерогенна, что вызывает трудности в определении основного пика на хроматограмме коммерческого образца. Testing is performed by HPLC gel filtration on a column with Superdex200 Increase 10/300 GL (GE) on an Alliance 2695 HPLC chromatography system, UV / Visible Detector 2487 (Waters). A buffer of 0.02 M NaH2P04 is used as the mobile phase; 0.3 m NaCl; pH = 7.0. The purified bN-GlcNAcase StrH preparation is introduced without dilution, commercial is diluted 2 times with the mobile phase before entering the system. The results of the analysis are presented in Fig.7. Testing the purified preparation of recombinant bN-GlcNAcase StrH in comparison with the commercial preparation of bN-GlcNAcase StrH is carried out in the same way. A comparison of the chromatographic profiles of the test samples shows (Fig. 8) that bN-GlcNAcase StrH StrH from MBC “Generium” is significantly more uniform in composition, while commercial bN-GlcNAcase StrH is more heterogeneous, which makes it difficult to determine the main peak in the chromatogram of a commercial sample .
Пример 8. Тестирование очищенных препаратов рекомбинантной b-N-GlcNAcase StrH в сравнении с коммерческим препаратом b-N-GlcNAcase на остаточную ДНК штамма продуцента. Example 8. Testing purified preparations of recombinant b-N-GlcNAcase StrH in comparison with the commercial preparation of b-N-GlcNAcase Strain on residual DNA of the producer strain.
Тестирование на остаточную ДНК штамма продуцента проводят методом количественного ПЦР в реальном времени, не более 20 пг/мг. Для дизайна количественной полимеразной цепной реакции (The quantitative polymerase chain reaction - QPCR) была выбрана система Taqman, в которой при размножении специфической последовательности ДНКа, зонд деградируется ДНКа-полимеразой. Набор праймеров и зонд для детекции ДНК штамма продуцента Е.соШ представлен в Таблице 6. Перед проведением тестирования образцы были очищены с использованием набора QIAamp DNA Blood Mini Kit (Qiagen). Конечный объём при элюции составил 50 мкл. В реакцию qPCR (пробу) добавляли 10 мкл образца. Количество повторов для каждого образца - 3. Testing for the residual DNA of the producer strain is carried out by real-time quantitative PCR, not more than 20 pg / mg. For the design of the quantitative polymerase chain reaction (QPCR), the Taqman system was chosen, in which, upon propagation of a specific DNA sequence, the probe is degraded by DNA polymerase. A set of primers and a probe for detecting the DNA of the producer E. coli strain are shown in Table 6. Before testing, the samples were purified using the QIAamp DNA Blood Mini Kit (Qiagen). The final volume during elution was 50 μl. 10 μl of sample was added to the qPCR reaction (sample). The number of repetitions for each sample is 3.
Количество ДНК в пробе менее 0,5 пг ниже предела достоверного обнаружения. Результаты анализа представлены в Таблице 7.
Таблица 6. Набор праймеров и зонд для детекции ДНК штамма продуцента E.coli.
The amount of DNA in the sample is less than 0.5 pg below the limit of reliable detection. The results of the analysis are presented in Table 7. Table 6. A set of primers and probe for the detection of DNA of a producer strain of E. coli.
Таблица 7. Результаты теста на остаточную ДНК штамма продуцента. Сравнение коммерческого препарата и полученных препаратов рекомбинантной b-N-GlcNAcase StrH. Table 7. Test results for residual DNA of the producer strain. Comparison of a commercial preparation and the resulting preparations of recombinant b-N-GlcNAcase StrH.
Claims
1. Способ получения рекомбинантной экзо-бета-N- ацетилглюкозаминидазы StrH в E.coli, отличающийся тем, что хроматографическую очистку проводят с использованием гексагистидинового тега, с применением последовательно стадий металлохелатной хроматографии, гель-фильтрации, анионобменной хроматографии, гидророфобной хроматографии, концентрирующей анионообменной хроматографии. 1. The method of obtaining recombinant exo-beta-N-acetylglucosaminidase StrH in E. coli, characterized in that the chromatographic purification is carried out using a hexahistidine tag, using sequential stages of metal chelate chromatography, gel filtration, anion exchange chromatography, hydrophobic chromatography, concentration anion exchange chromatography .
2. Способ по п.1, где металлхелатную хроматографию проводят на 2. The method according to claim 1, where the metal chelate chromatography is carried out on
IMAC Sepharose 6 FF. IMAC Sepharose 6 FF.
3. Способ по п.1, где промывка на стадии металлхелатной хроматографии проводится кислым буфером с высокой ионной силой. 3. The method according to claim 1, where the washing at the stage of metal chelate chromatography is carried out with acidic buffer with high ionic strength.
4. Способ по п.1, где гель-фильтрацию проводят на Blue Sepharose в режиме проскока, 4. The method according to claim 1, where the gel filtration is carried out on Blue Sepharose in a slip mode,
5. Способ по п.1, где анионобменную хроматографию проводят на Toyopearl Giga Cap Q-650M. 5. The method according to claim 1, where the anion exchange chromatography is performed on Toyopearl Giga Cap Q-650M.
6. Способ по п.1, где гидрофобную хроматографию проводят на сорбенте Capto Butyl. 6. The method according to claim 1, where the hydrophobic chromatography is performed on a Capto Butyl sorbent.
7. Способ по п.1, где анионообменную хроматографию проводят на 7. The method according to claim 1, where the anion exchange chromatography is carried out on
Q Sepharose FF. Q Sepharose FF.
8. Способ по п.1, где дополнительно проводят концентрирование белка ультрафильтрацией с использованием мембраны с отсечением 50 кДа и гель - фильтрацию на Superdex 200. 8. The method according to claim 1, where the protein is further concentrated by ultrafiltration using a membrane with a cut-off of 50 kDa and gel filtration on Superdex 200.
9. Способ по п.1, где аминокислотная последовательность рекомбинантной экзо-бета-Г -ацетилглюкозаминидазы StrH с гексагистидиновым тагом идентична последовательности SEQ ID NO 2.
9. The method according to claim 1, where the amino acid sequence of the recombinant exo-beta-G-acetylglucosaminidase StrH with hexahistidine tag is identical to the sequence of SEQ ID NO 2.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| RU2018103832 | 2018-02-01 | ||
| RU2018103832A RU2693660C1 (en) | 2018-02-01 | 2018-02-01 | METHOD OF PRODUCING RECOMBINANT BETA-N-ACETYLGLUCOSAMINIDASE StrH FROM STREPTOCOCCUS PNEUMONIAE |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO2019151901A1 true WO2019151901A1 (en) | 2019-08-08 |
Family
ID=67252311
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/RU2019/000045 WO2019151901A1 (en) | 2018-02-01 | 2019-01-25 | Method of producing recombinant beta-n-acetylglucosaminidase strh from streptococcus pneumoniae |
Country Status (2)
| Country | Link |
|---|---|
| RU (1) | RU2693660C1 (en) |
| WO (1) | WO2019151901A1 (en) |
-
2018
- 2018-02-01 RU RU2018103832A patent/RU2693660C1/en active
-
2019
- 2019-01-25 WO PCT/RU2019/000045 patent/WO2019151901A1/en active Application Filing
Non-Patent Citations (4)
| Title |
|---|
| ARORA I.: "Chromatographic methods for the purification of monoclonal antibodies and their alternatives: A review", IJETAE, vol. 3, no. 10, 2013, pages 475 - 481, XP055628474 * |
| DATABASE Protein 9 February 2016 (2016-02-09), "beta-N-acetylhexosaminidase [Streptococcus pneumoniae]", XP055628481, retrieved from ncbi Database accession no. CWM34019.1 * |
| KUBOTA T. ET AL.: "Molecular characterization of an intracellular beta-N- acetylglucosaminidase involved in the chitin degradation system of Streptomyces thermoviolaceus OPC-520", BIOSCIENCE, BIOTECHNOLOGY, AND BIOCHEMISTRY, vol. 68, no. 6, January 2004 (2004-01-01), pages 1306 - 1314, XP055628477 * |
| PLUVINAGE B. ET AL.: "Conformational analysis of StrH, the surface-attached exo-beta- dN-acetylglucosaminidase from Streptococcus pneumoniae", JOURNAL OF MOLECULAR BIOLOGY, vol. 425, no. 2, January 2013 (2013-01-01), pages 334 - 349, XP055628473 * |
Also Published As
| Publication number | Publication date |
|---|---|
| RU2693660C1 (en) | 2019-07-03 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| WO2007009351A1 (en) | A method of secretion expression of lysostaphin in escherichia coli at high level | |
| JP2011172571A (en) | Recombinant protein comprising starch binding domain and use thereof | |
| Stojković et al. | Coliphage N4 N-acetylmuramidase defines a new family of murein hydrolases | |
| CN114015708B (en) | Deep sea bacteria-derived alpha-glucosidase QsGH13 and encoding gene and application thereof | |
| CA2909440C (en) | A method of production of rare disaccharides | |
| JP7105761B2 (en) | Methods for Affinity Purification of Recombinant Proteins Based on the Lectin Activity of Galectin CRDs | |
| CN109022405A (en) | A kind of Cold tolerance algin catenase AlgA5 and its application | |
| RU2693660C1 (en) | METHOD OF PRODUCING RECOMBINANT BETA-N-ACETYLGLUCOSAMINIDASE StrH FROM STREPTOCOCCUS PNEUMONIAE | |
| CN116949004A (en) | Transpeptidase Sortase A and preparation method and application thereof | |
| CN114645033B (en) | Nucleoside triphosphate hydrolase and purification method and application thereof | |
| CN116694584A (en) | Preparation method of T4DNA ligase | |
| Tielker et al. | Lectin-based affinity tag for one-step protein purification | |
| RU2698460C1 (en) | Method of producing recombinant β-1,4-galactosidase bgaa from streptococcus pneumoniae | |
| EP1432822B1 (en) | Method for recovering and analyzing a cellular component of cultured cells without having to harvest the cells first | |
| RU2698774C1 (en) | METHOD OF PRODUCING RECOMBINANT NEURAMINIDASE NanH FROM CLOSTRIDIUM PERERINGENS | |
| KR101673195B1 (en) | The Recombinant Sialic Acid-Specific Binding Lectin, PSL1b, from the Mushroom Polyporus squamosus | |
| JP6993637B2 (en) | Endoglycosidase that specifically cleaves fucose-containing sugar chains | |
| US20120052533A1 (en) | 4s-iota-carrageenan sulfatase and use thereof to obtain alpha-carrageenan | |
| US8669089B2 (en) | Ulvan lyase, method for manufacturing same, and uses thereof | |
| CN112391367A (en) | Preparation method of Cas9 protein for gene editing of human primary cells | |
| JP4781851B2 (en) | Novel carbohydrate hydrolase that hydrolyzes αN-acetylglucosaminyl linkage | |
| US7662606B1 (en) | β-agarase isolated from Thalassomonas agarivorans, preparation process and uses thereof | |
| JPWO2020040257A1 (en) | Enzyme synthesis method of β-glycoside of lacto-N-biose I or galacto-N-biose using modified phosphorylase | |
| US11248217B2 (en) | Engineered carbohydrate-active enzymes for glycan polymers synthesis | |
| CN113088505B (en) | Application of polysaccharide lyase coding gene 04147 in preparation of recombinant peach gum polysaccharide hydrolase |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| 121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 19746830 Country of ref document: EP Kind code of ref document: A1 |
|
| NENP | Non-entry into the national phase |
Ref country code: DE |
|
| 122 | Ep: pct application non-entry in european phase |
Ref document number: 19746830 Country of ref document: EP Kind code of ref document: A1 |