WO2024110932A1 - The fgf-2 polypeptides with improved stability, the process for preparing the fgf-2 polypeptides and use thereof - Google Patents
The fgf-2 polypeptides with improved stability, the process for preparing the fgf-2 polypeptides and use thereof Download PDFInfo
- Publication number
- WO2024110932A1 WO2024110932A1 PCT/IB2023/061877 IB2023061877W WO2024110932A1 WO 2024110932 A1 WO2024110932 A1 WO 2024110932A1 IB 2023061877 W IB2023061877 W IB 2023061877W WO 2024110932 A1 WO2024110932 A1 WO 2024110932A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- fgf
- seq
- polypeptide
- polypeptide according
- thermostable
- Prior art date
Links
- 229920001184 polypeptide Polymers 0.000 title claims abstract description 21
- 102000004196 processed proteins & peptides Human genes 0.000 title claims abstract description 21
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 21
- 238000004519 manufacturing process Methods 0.000 title description 13
- 101150019331 FGF2 gene Proteins 0.000 title description 6
- 102000003974 Fibroblast growth factor 2 Human genes 0.000 claims abstract description 244
- 108090000379 Fibroblast growth factor 2 Proteins 0.000 claims abstract description 244
- 238000000034 method Methods 0.000 claims abstract description 19
- 230000008569 process Effects 0.000 claims abstract description 13
- 239000002537 cosmetic Substances 0.000 claims abstract description 7
- 150000001413 amino acids Chemical group 0.000 claims description 34
- 229940024606 amino acid Drugs 0.000 claims description 20
- 238000006467 substitution reaction Methods 0.000 claims description 19
- 238000004113 cell culture Methods 0.000 claims description 16
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 claims description 14
- 101100281005 Bos taurus FGF2 gene Proteins 0.000 claims description 12
- 229960000310 isoleucine Drugs 0.000 claims description 10
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 claims description 10
- 102200031779 rs3204853 Human genes 0.000 claims description 8
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical group C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 claims description 7
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 claims description 7
- 238000002844 melting Methods 0.000 claims description 7
- 230000008018 melting Effects 0.000 claims description 7
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical group CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 claims description 6
- 102220621903 N-acetylglucosamine-6-sulfatase_S94I_mutation Human genes 0.000 claims description 6
- 239000001963 growth medium Substances 0.000 claims description 6
- 102220004441 rs121918684 Human genes 0.000 claims description 6
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Chemical group CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 claims description 5
- 238000012217 deletion Methods 0.000 claims description 5
- 230000037430 deletion Effects 0.000 claims description 5
- 238000003306 harvesting Methods 0.000 claims description 5
- 239000004474 valine Chemical group 0.000 claims description 5
- 125000000741 isoleucyl group Chemical group [H]N([H])C(C(C([H])([H])[H])C([H])([H])C([H])([H])[H])C(=O)O* 0.000 claims description 4
- 235000013622 meat product Nutrition 0.000 claims description 4
- 125000002987 valine group Chemical group [H]N([H])C([H])(C(*)=O)C([H])(C([H])([H])[H])C([H])([H])[H] 0.000 claims description 4
- 238000012806 monitoring device Methods 0.000 claims description 3
- 102200104421 rs104894658 Human genes 0.000 claims description 2
- 102200008028 rs121912294 Human genes 0.000 claims description 2
- 102220058659 rs767802663 Human genes 0.000 claims description 2
- 102220158997 rs886047688 Human genes 0.000 claims description 2
- 230000000063 preceeding effect Effects 0.000 claims 1
- 230000004071 biological effect Effects 0.000 abstract description 17
- 238000011160 research Methods 0.000 abstract description 8
- 235000013372 meat Nutrition 0.000 abstract description 7
- 238000002360 preparation method Methods 0.000 abstract description 6
- 238000012604 3D cell culture Methods 0.000 abstract description 5
- 210000002220 organoid Anatomy 0.000 abstract description 5
- 239000003814 drug Substances 0.000 abstract description 4
- 210000004027 cell Anatomy 0.000 description 57
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 36
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 24
- 238000004458 analytical method Methods 0.000 description 15
- 239000000047 product Substances 0.000 description 15
- 239000000872 buffer Substances 0.000 description 14
- 108090000623 proteins and genes Proteins 0.000 description 14
- 102000004169 proteins and genes Human genes 0.000 description 14
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 13
- 239000011780 sodium chloride Substances 0.000 description 12
- 238000002835 absorbance Methods 0.000 description 11
- 238000010828 elution Methods 0.000 description 11
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 9
- 101001052035 Homo sapiens Fibroblast growth factor 2 Proteins 0.000 description 8
- 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 8
- 102000018233 Fibroblast Growth Factor Human genes 0.000 description 7
- 108050007372 Fibroblast Growth Factor Proteins 0.000 description 7
- 101000878176 Homo sapiens Fibroblast growth factor 13 Proteins 0.000 description 7
- 150000001875 compounds Chemical class 0.000 description 7
- 229930027917 kanamycin Natural products 0.000 description 7
- 229960000318 kanamycin Drugs 0.000 description 7
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 7
- 229930182823 kanamycin A Natural products 0.000 description 7
- 238000000746 purification Methods 0.000 description 7
- 239000000243 solution Substances 0.000 description 7
- 241000588724 Escherichia coli Species 0.000 description 6
- 108091008794 FGF receptors Proteins 0.000 description 6
- 239000006137 Luria-Bertani broth Substances 0.000 description 6
- 229920000669 heparin Polymers 0.000 description 6
- 229960002897 heparin Drugs 0.000 description 6
- 230000035772 mutation Effects 0.000 description 6
- 238000011296 nano differential scanning fluorimetry Methods 0.000 description 6
- 239000008188 pellet Substances 0.000 description 6
- 239000007995 HEPES buffer Substances 0.000 description 5
- 230000001580 bacterial effect Effects 0.000 description 5
- 238000005119 centrifugation Methods 0.000 description 5
- 238000006731 degradation reaction Methods 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 239000000499 gel Substances 0.000 description 5
- 230000007774 longterm Effects 0.000 description 5
- 239000006166 lysate Substances 0.000 description 5
- 238000000527 sonication Methods 0.000 description 5
- 238000003860 storage Methods 0.000 description 5
- 102000044168 Fibroblast Growth Factor Receptor Human genes 0.000 description 4
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 description 4
- 239000003242 anti bacterial agent Substances 0.000 description 4
- 230000010261 cell growth Effects 0.000 description 4
- 230000003993 interaction Effects 0.000 description 4
- YFVGRULMIQXYNE-UHFFFAOYSA-M lithium;dodecyl sulfate Chemical compound [Li+].CCCCCCCCCCCCOS([O-])(=O)=O YFVGRULMIQXYNE-UHFFFAOYSA-M 0.000 description 4
- 239000013612 plasmid Substances 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 238000005406 washing Methods 0.000 description 4
- 229920001817 Agar Polymers 0.000 description 3
- 239000008272 agar Substances 0.000 description 3
- 229940088710 antibiotic agent Drugs 0.000 description 3
- 230000015556 catabolic process Effects 0.000 description 3
- 230000003247 decreasing effect Effects 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 239000010931 gold Substances 0.000 description 3
- 229910052737 gold Inorganic materials 0.000 description 3
- 230000006698 induction Effects 0.000 description 3
- 238000010829 isocratic elution Methods 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 238000010899 nucleation Methods 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 241000283690 Bos taurus Species 0.000 description 2
- 108020004705 Codon Proteins 0.000 description 2
- 241000672609 Escherichia coli BL21 Species 0.000 description 2
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 description 2
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 description 2
- 229920002971 Heparan sulfate Polymers 0.000 description 2
- 108010022901 Heparin Lyase Proteins 0.000 description 2
- 241000282414 Homo sapiens Species 0.000 description 2
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 2
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 2
- 102000016943 Muramidase Human genes 0.000 description 2
- 108010014251 Muramidase Proteins 0.000 description 2
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 description 2
- 102000001708 Protein Isoforms Human genes 0.000 description 2
- 108010029485 Protein Isoforms Proteins 0.000 description 2
- 229920002684 Sepharose Polymers 0.000 description 2
- 239000012736 aqueous medium Substances 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 239000007640 basal medium Substances 0.000 description 2
- 230000003115 biocidal effect Effects 0.000 description 2
- 238000011138 biotechnological process Methods 0.000 description 2
- 230000005754 cellular signaling Effects 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 239000013604 expression vector Substances 0.000 description 2
- 210000002744 extracellular matrix Anatomy 0.000 description 2
- 102000052178 fibroblast growth factor receptor activity proteins Human genes 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 230000001939 inductive effect Effects 0.000 description 2
- 230000010354 integration Effects 0.000 description 2
- 230000003834 intracellular effect Effects 0.000 description 2
- 239000004325 lysozyme Substances 0.000 description 2
- 229960000274 lysozyme Drugs 0.000 description 2
- 235000010335 lysozyme Nutrition 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 239000002773 nucleotide Substances 0.000 description 2
- 125000003729 nucleotide group Chemical group 0.000 description 2
- 230000004845 protein aggregation Effects 0.000 description 2
- 230000017854 proteolysis Effects 0.000 description 2
- 102000005962 receptors Human genes 0.000 description 2
- 108020003175 receptors Proteins 0.000 description 2
- 239000000523 sample Substances 0.000 description 2
- 239000012723 sample buffer Substances 0.000 description 2
- 230000011664 signaling Effects 0.000 description 2
- 238000001542 size-exclusion chromatography Methods 0.000 description 2
- 230000000087 stabilizing effect Effects 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 238000010257 thawing Methods 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 239000004475 Arginine Substances 0.000 description 1
- 102220534634 Aryl hydrocarbon receptor nuclear translocator-like protein 1_R11I_mutation Human genes 0.000 description 1
- 101001051969 Bos taurus Fibroblast growth factor 2 Proteins 0.000 description 1
- 102000014914 Carrier Proteins Human genes 0.000 description 1
- 102000000844 Cell Surface Receptors Human genes 0.000 description 1
- 108010001857 Cell Surface Receptors Proteins 0.000 description 1
- 102000003971 Fibroblast Growth Factor 1 Human genes 0.000 description 1
- 108090000386 Fibroblast Growth Factor 1 Proteins 0.000 description 1
- 102000003886 Glycoproteins Human genes 0.000 description 1
- 108090000288 Glycoproteins Proteins 0.000 description 1
- 229920001499 Heparinoid Polymers 0.000 description 1
- 102220565922 Histatin-3_R31I_mutation Human genes 0.000 description 1
- 101100281008 Homo sapiens FGF2 gene Proteins 0.000 description 1
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 description 1
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 108091000080 Phosphotransferase Proteins 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 229940124158 Protease/peptidase inhibitor Drugs 0.000 description 1
- 102000004022 Protein-Tyrosine Kinases Human genes 0.000 description 1
- 108090000412 Protein-Tyrosine Kinases Proteins 0.000 description 1
- 102000016611 Proteoglycans Human genes 0.000 description 1
- 108010067787 Proteoglycans Proteins 0.000 description 1
- 241000283984 Rodentia Species 0.000 description 1
- 239000012505 Superdex™ Substances 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 239000008186 active pharmaceutical agent Substances 0.000 description 1
- 102000035181 adaptor proteins Human genes 0.000 description 1
- 108091005764 adaptor proteins Proteins 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- 230000002052 anaphylactic effect Effects 0.000 description 1
- 210000004102 animal cell Anatomy 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
- 125000000637 arginyl group Chemical group N[C@@H](CCCNC(N)=N)C(=O)* 0.000 description 1
- 108091008324 binding proteins Proteins 0.000 description 1
- 238000013452 biotechnological production Methods 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 238000005277 cation exchange chromatography Methods 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 238000007385 chemical modification Methods 0.000 description 1
- 238000004891 communication Methods 0.000 description 1
- 238000010668 complexation reaction Methods 0.000 description 1
- 238000005094 computer simulation Methods 0.000 description 1
- 239000006071 cream Substances 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 239000007857 degradation product Substances 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 235000014113 dietary fatty acids Nutrition 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 239000000539 dimer Substances 0.000 description 1
- 238000006471 dimerization reaction Methods 0.000 description 1
- 229930195729 fatty acid Natural products 0.000 description 1
- 239000000194 fatty acid Substances 0.000 description 1
- 150000004665 fatty acids Chemical class 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 238000001641 gel filtration chromatography Methods 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 239000003102 growth factor Substances 0.000 description 1
- 239000002554 heparinoid Substances 0.000 description 1
- 229940025770 heparinoids Drugs 0.000 description 1
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 1
- 125000000487 histidyl group Chemical group [H]N([H])C(C(=O)O*)C([H])([H])C1=C([H])N([H])C([H])=N1 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
- 210000000936 intestine Anatomy 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- 125000001909 leucine group Chemical group [H]N(*)C(C(*)=O)C([H])([H])C(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- 239000003446 ligand Substances 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 239000006210 lotion Substances 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 229910001629 magnesium chloride Inorganic materials 0.000 description 1
- 210000004962 mammalian cell Anatomy 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 229940126601 medicinal product Drugs 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 239000011785 micronutrient Substances 0.000 description 1
- 235000013369 micronutrients Nutrition 0.000 description 1
- 239000011707 mineral Substances 0.000 description 1
- 238000003032 molecular docking Methods 0.000 description 1
- 230000009456 molecular mechanism Effects 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 1
- 239000000546 pharmaceutical excipient Substances 0.000 description 1
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 1
- COLNVLDHVKWLRT-QMMMGPOBSA-N phenylalanine group Chemical group N[C@@H](CC1=CC=CC=C1)C(=O)O COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 1
- 102000020233 phosphotransferase Human genes 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 230000004481 post-translational protein modification Effects 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 230000004853 protein function Effects 0.000 description 1
- 230000006884 regulation of angiogenesis Effects 0.000 description 1
- 230000003716 rejuvenation Effects 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000002864 sequence alignment Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 102000034285 signal transducing proteins Human genes 0.000 description 1
- 108091006024 signal transducing proteins Proteins 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 230000006641 stabilisation Effects 0.000 description 1
- 238000011105 stabilization Methods 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 235000000346 sugar Nutrition 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 230000009469 supplementation Effects 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 238000013268 sustained release Methods 0.000 description 1
- 239000012730 sustained-release form Substances 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 125000001493 tyrosinyl group Chemical group [H]OC1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 description 1
- 230000000007 visual effect Effects 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 230000037314 wound repair Effects 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/475—Growth factors; Growth regulators
- C07K14/50—Fibroblast growth factor [FGF]
- C07K14/503—Fibroblast growth factor [FGF] basic FGF [bFGF]
Definitions
- the invention relates to Fibroblast Growth Factor 2 (FGF-2) polypeptide with improved stability, especially thermal stability, compared to the wild-type of FGF-2 and the use thereof in research and industrial applications, such as biotechnological research, medicine, pharmaceutical industry, cosmetics, clean meat industry, generation of organoids and 3D cell culture models and other related applications.
- FGFs Fibroblast growth factors
- FGFs are a family of cell signaling proteins. They are involved in a variety of processes, especially as key elements for normal development in animal cells.
- FGF receptors Fibroblast growth factors that signal through FGF receptors (FGFRs) regulate fundamental developmental pathways, including the regulation of angiogenesis and wound repair. FGFRs are expressed on many different cell types and regulate key cell processes, such as proliferation, differentiation and survival, which make FGF signaling susceptible to subversion by cancer cells. FGFs are secreted glycoproteins that are generally readily sequestered to the extracellular matrix, as well as the cell surface, by heparan sulphate proteoglycans (HPSGs).
- HPSGs heparan sulphate proteoglycans
- FGFs are released from the extracellular matrix by heparinases, proteases or specific FGF-binding proteins, and the liberated FGFs subsequently bind to cell surface HPSGs.
- Cell surface HPSGs also stabilize the FGF ligand–receptor interaction, forming a ternary complex with FGFR.
- FGF receptors signal as dimers, and ligand-dependent dimerization leads to a conformational shift in receptor structure that activates the intracellular kinase domain, resulting in intermolecular transphosphorylation of the tyrosine kinase domains and intracellular tail.
- the human FGF-2 gene encodes not one protein, but a complex set of isoforms.
- the secreted isoform is a single-chain, non-glycosylated polypeptide with 154 amino acids.
- the amino acid sequence of human FGF-2 is 99% homologous to that of bovine FGF-2 and has high homology with ovine and rodent FGF-2, suggesting strong sequence conservation for structure and function.
- the stability of FGF-2 is widely accepted to be a major concern in the development of useful medicinal products or serum replacement in biotechnological research.
- the reconstituted FGFs solutions are stable for only about a week at 4 °C and are recommended to be used within 24 h when at ambient temperatures (around 25 °C). Nevertheless, a 50% loss of functionality of FGF-2 solutions at a concentration of 72 ⁇ g/mL was observed after just 4 min at 25 °C.
- One of the challenges is to preserve the biological activity of FGF-2.
- FGF-2 farnesoid GF-2
- approaches to maintain the stability and biological activity of FGF-2 include modulating ionic interactions in solution and chemical modifications of FGF-2.
- excipients to aqueous solutions of FGF-2 is among the simplest methods for FGF-2 stabilization by way of modulating ionic interactions in solution.
- Common strategies include the complexation of FGF-2 with its endogenous stabilizer, heparin or heparin-like polymers, or polycations. Ionic interactions between FGF-2 and the additives reduce the structural energy at the heparin-binding site, stabilize the FGF-2 native conformation and prolong its bioactivity in aqueous media.
- Mutant variants of the FGF-2 protein have been developed by aligning the wild-type FGF-2 protein sequence with stabilized FGF-1 mutant sequences, or by combining several individual stabilizing mutations identified by other investigators in the field. The identification of useful point mutations has been aided by computer modeling. Sequences have been identified that are important for protein functionality and contribute the greatest amount to structural free energy. Following an analysis of sequence conservation, mutations in regions, which might compromise protein function, were avoided. Conversely, mutations that were likely to result in a decrease in protein free energy were promoted. The obtained FGF-2 mutants showed the lowest energy and had increased functional half-life. Despite some advances within the state of the art, there is still need to further improve the stability of the FGF-2 product while maintaining its biological activity.
- the object of the invention is to prepare an FGF-2 variant with improved stability and to find an efficient, scalable and economically advantageous process for the production of FGF-2 with high yields.
- the disadvantages of the solutions, according to the state of the art, are solved by the present invention that provides a thermostable polypeptide that possesses FGF-2 activity and has increased stability while maintaining biological activity compared to the wild-type FGF-2.
- the product is truncated thermostabilized FGF-2 polypeptide with amino acid substitutions characterized by sequence SEQ ID NO: 3.
- the FGF-2 polypeptide with SEQ ID NO: 3 has 80.6% sequence identity to Homo sapiens FGF-2 (SEQ ID NO: 1) and 81.3% sequence identity to Bos taurus FGF-2 (SEQ ID NO: 2).
- the product is a thermostabilized FGF-2 polypeptide with amino acid substitutions characterized by sequence SEQ ID NO: 4.
- the objects of the present invention are also other FGF-2 polypeptides that are derived from the amino acid sequence of SEQ ID NO: 3, for example FGF-2 polypeptides having at least 90% sequence identity, or at least 93% sequence identity, or at least 95% sequence identity to the sequence of the truncated thermostabilized FGF-2 polypeptide (SEQ ID NO: 3), FGF-2 polypeptides characterized by SEQ ID NO: 5-12 or SEQ ID NO:21 (which contains substitution L63Y), or other FGF-2 polypeptides according to the description below.
- FGF-2 polypeptides having at least 90% sequence identity, or at least 93% sequence identity, or at least 95% sequence identity to the sequence of the truncated thermostabilized FGF-2 polypeptide (SEQ ID NO: 3), FGF-2 polypeptides characterized by SEQ ID NO: 5-12 or SEQ ID NO:21 (which contains substitution L63Y), or other FGF-2 polypeptides according to the description below.
- the objects of the present invention are also other FGF-2 polypeptides that are derived from the amino acid sequence of SEQ ID NO: 4, for example FGF-2 polypeptides having at least 90% sequence identity, or at least 93% sequence identity, or at least 95% sequence identity to the sequence of the thermostabilized FGF-2 polypeptide (SEQ ID NO: 4), for example FGF-2 polypeptides characterized by SEQ ID NO: 13 to 20.
- the objects of the present invention are also dimeric variants of the FGF-2 polypeptide comprising a linker, for example GSS linker or SUMO linker.
- the objects of the present invention are also dimeric variants of the FGF-2 polypeptide characterized by SEQ ID NO: 22-27.
- the dimeric variants of the FGF-2 polypeptide may comprise two connected sequences characterized by SEQ ID NO: 3 or two connected sequences having at least 90%, or 93%, or 95% sequence identity to the SEQ ID NO: 3.
- the linker for connecting two sequences may be for example a GSS linker (e.g. 6xGSS or 10xGSS) as used in dimeric FGF-2 polypeptides characterized by SEQ ID NO: 22 – 23, or SUMO linkers characterized by SEQ ID NO: 28-31 as used in dimeric FGF-2 polypeptides characterized by SEQ ID NO: 24 - 27.
- the invention also provides the process for preparation of the FGF-2 polypeptides and the use thereof in the pharmaceutical industry, cosmetics, clean meat industry generation of organoids and 3D cell culture models and other related applications.
- the increased stability while maintaining biological activity compared to the Homo sapiens FGF-2 has been proven by experiments.
- the biological activity has been tested by cell growth experiments, the biological effects of commercially available FGF-2 and FGF-2 products according to the invention have been compared.
- the thermal stability of the FGF-2 polypeptides using nano differential scanning fluorimetry (nanoDSF) method, and the effect of long-term storage on product stability have been investigated. Brief description of the several views of the drawings Fig.
- Fig.5 Thermostability of truncated thermostabilized FGF-2 characterized by SEQ ID NO: 3 analyzed by nanoDSF. Changes in tryptophan emission at 330 and 350 nm were monitored, and the ratio 330/350 nm was plotted against the temperature. Representative thermal unfolding curve (top) and its first derivative analysis (bottom) Fig. 6 - Stability of truncated thermostabilized FGF-2 characterized by SEQ ID NO: 3 after storage at 4°C for 30 days Fig.
- thermostabilized FGF-2 characterized by SEQ ID NO: 3 after freezing-thawing cycle
- Fig. 8 The results from the cultivation of cells, when commercially available FGF-2 and truncated thermostabilized FGF-2 characterized by SEQ ID NO: 3 were compared
- Fig. 9 SDS-PAGE analysis of the expression of thermostabilized FGF-2 polypeptides.
- thermostable polypeptides that possess FGF-2 activity and have increased stability compared to the wild-type FGF-2, while maintaining the biological activity.
- the invention also provides the process for preparation of the FGF-2 polypeptides and use thereof.
- the product is a thermostable FGF-2 polypeptide derived from Bos taurus FGF-2 (SEQ ID NO: 2) comprising at least one of the amino acids substitutions R31L, V52T, E54D, H59F, L92Y, S94I, C96N, S109E, S121P.
- the product is a truncated thermostabilized FGF-2 polypeptide characterized by sequence SEQ ID NO: 3, Fig. 1.
- the sequence SEQ ID NO: 3 is derived from the Bos taurus FGF-2 polypeptide (SEQ ID NO: 2), with the following modifications: deletion of amino acids 1-20 at the N-terminus, and nine amino acids substitutions, specifically R31L, V52T, E54D, H59F, L92Y, S94I, C96N, S109E, S121P.
- the Bos taurus FGF-2 SEQ ID NO: 2
- the truncated thermostabilized FGF-2 polypeptide SEQ ID NO: 3 have the modification S137P.
- the FGF-2 polypeptides may be derived from the Bos taurus FGF-2 polypeptide (SEQ ID NO: 2), with deletion of amino acids 1-15 to 1-22 at the N- terminus. These polypeptides may comprise at least one of amino acids substitutions: R31L, V52T, E54D, H59F, L92Y, S94I, C96N, S109E, or S121P relative to SEQ ID NO: 2.
- thermostabilized FGF-2 polypeptide (SEQ ID NO: 3), according to the present invention, has 80.6% sequence identity to Homo sapiens FGF-2 (SEQ ID NO: 1) and 81.3% sequence identity to Bos taurus FGF-2 (SEQ ID NO: 2).
- the product is a thermostabilized FGF-2 polypeptide characterized by sequence SEQ ID NO: 4, Fig. 1. Nucleotide sequence encoding the thermostabilized FGF-2 was cloned into the pET30a(+) expression vector.
- thermostabilized FGF-2 polypeptide (SEQ ID NO: 4), according to the present invention, has 93.5% sequence identity to Homo sapiens FGF-2 a sequence (SEQ ID NO: 1) and 94.2% sequence identity to Bos taurus FGF-2 (a sequence SEQ ID NO: 2).
- the objects of the present invention are also other FGF-2 polypeptides that are derived from the amino acid sequence of SEQ ID NO: 3, for example FGF-2 polypeptides having at least 90% sequence identity, or at least 93% sequence identity, or at least 95% sequence identity to the sequence of the truncated thermostabilized FGF-2 polypeptide (SEQ ID NO: 3).
- the objects of the present invention are also FGF-2 polypeptides derived from the amino acid sequence of SEQ ID NO: 3 comprising at least one of amino acids substitutions: R11L, V32T, E34D, H39F, L72Y, S74I, C76N, S89E, or S101P.
- the objects of the present invention are also FGF-2 polypeptides characterized by SEQ ID NO: 5-12 or SEQ ID NO: 21 (which contains substitution L63Y), or other FGF-2 polypeptides according to the description below.
- the objects of the present invention are also other FGF-2 polypeptides that are derived from the amino acid sequence of SEQ ID NO: 4, for example FGF-2 polypeptides having at least 90% sequence identity, or at least 93% sequence identity, or at least 95% sequence identity to the sequence of the thermostabilized FGF-2 polypeptide (SEQ ID NO: 4), for example FGF- 2 polypeptides characterized by SEQ ID NO: 13 to 20.
- the thermostable FGF-2 polypeptide derived from the amino acid sequence of SEQ ID NO: 4 may comprise at least one of the amino acids substitutions R31L, V52T, E54D, H59F, L92Y, S94I, C96N, S109E, or S121P.
- the products are dimeric variants of the FGF-2 polypeptide comprising a linker, for example GSS linker or SUMO linker.
- the objects of the present invention are also dimeric variants of the FGF-2 polypeptide characterized by SEQ ID NO: 22-27.
- the dimeric variants of the FGF-2 polypeptide according to the invention may comprise two connected sequences characterized by SEQ ID NO: 3 or two connected sequences having at least 90%, or 93%, or 95% sequence identity to the SEQ ID NO: 3.
- the linker for connecting two sequences may be for example a GSS linker (6xGSS or 10xGSS) as used in dimeric FGF-2 polypeptides characterized by SEQ ID NO: 22 – 23, or SUMO linkers characterized by SEQ ID NO: 28-31 as used in dimeric FGF-2 polypeptides characterized by SEQ ID NO: 24 - 27.
- the thermostabilized FGF-2 polypeptide may comprise amino acid substitution R31L (arginine at position 31 substituted by leucine) and H59F (histidine at position 59 substituted by phenylalanine) in the amino acid sequence of the Bos taurus FGF- 2 polypeptide (SEQ ID NO: 2).
- thermo-stabilized FGF-2 polypeptide may comprise another amino acid substitutions than R31L and H59F substitution in SEQ ID NO: 2 or SEQ ID NO: 4.
- thermostabilized FGF-2 polypeptide may comprise R31 substitution with any appropriate amino acid, for example with isoleucine or valine, and H59 substitution with any appropriate amino acid, for example with tryptophan or isoleucine.
- the object of the present invention is also FGF-2 polypeptides (SEQ ID NO: 5-12) that are derived from the amino acid sequence of SEQ ID NO: 3 and comprising the following two residues: (i) isoleucine, valine at position 11 of SEQ ID NO: 3 (i.e., R11I, R11V) (ii) tryptophan, isoleucine at position 39 of SEQ ID NO: 3 (i.e., H39W, H39I).
- the object of the present invention is also FGF-2 polypeptides (SEQ ID NO: 13-20) that are derived from the amino acid sequence of SEQ ID NO: 4 and comprising the following two residues: (i) isoleucine, valine at position 31 of SEQ ID NO: 4 (i.e., R31I, R31V) (ii) tryptophan, isoleucine at position 59 of SEQ ID NO: 4 (i.e., H59W, H59I).
- the object of the present invention is also thermostabilized FGF-2 polypeptides with the melting temperature (Tm) higher than or equal to 55 °C, or higher than or equal to 65 °C, or higher than or equal to 68 °C.
- thermostabilized FGF-2 polypeptides may be in the range of 65 to 80 °C, or in the range of 67 to 75 °C, or in the range of 68 to 72 °C.
- the process according to the invention may consist of the steps: - Transformation of the competent E. coli cells with the plasmid DNA carrying the relevant FGF-2 sequence - Selection and screening of the transformed cells - Cultivation of the transformed cells - Induction of FGF-2 production - FGF-2 detection - FGF-2 isolation and purification - FGF-2 characterization.
- the bacterial expression host system for example E.
- coli bacterial expression host system may be used for the production of FGF-2 polypeptides due to its low cost, well-known biochemistry and genetics, rapid growth, and good productivity.
- a bacterial expression system is ideal for FGF-2 production as FGF-2 is a relatively small and single-domain protein with a compact fold.
- crystal structures of FGF-FGFR-heparin complexes and molecular mechanisms of the action of FGF proteins suggest that no post-translational modifications or cofactors of FGF proteins are required for their proper function.
- FGF proteins purified from E. coli showed biological activity. Therefore, the bacterial expression host system appears to be ideal for the large-scale industrial production of FGF-2. The presence of rare codons may be addressed by using codon optimization.
- the strains used for the production of FGF-2 polypeptides according to the invention may be E. coli strains, for example, BL21, BL-21-Gold or BL21- CodonPlus RIPL.
- the competent cells for the production of FGF-2 polypeptides are transformed with the plasmid DNA carrying the relevant FGF-2 sequence, encoding for example the FGF- 2 polypeptide as defined by sequence SEQ ID NO: 3 or SEQ ID NO: 4, or any other sequences according to the invention, e.g. SEQ ID NO: 5 to 27.
- the transformed cells are plated onto solid selection media, for example suspension of cells may be plated on LB (Luria-Bertani) agar plates supplemented with antibiotics.
- the colonies are later, for example next day, picked and transferred into liquid selection media, for example LB broth supplemented with antibiotics.
- liquid selection media for example LB broth supplemented with antibiotics.
- the cells are grown for several hours, for example overnight.
- the culture may be then placed into fresh liquid selection medium, for example LB broth supplemented with antibiotics, and incubated at temperature in the range of 15 to 45 °C, or in the range of 20 to 40 °C, or in the range of 30 to 37 °C.
- an antibiotic may be used for example kanamycin, or any other appropriate antibiotic.
- an inducing agent for example isopropyl ⁇ -D-thiogalactoside (IPTG), or any other appropriate inducing agent may be added to cell cultures for induction of FGF-2 production.
- IPTG isopropyl ⁇ -D-thiogalactoside
- the concentration of IPTG may be in the range of 0.001 to 10 mM, or in the range of 0.01 to 1 mM, or in the range of 0.05 to 0.5 mM.
- the cell cultures are incubated at a temperature in the range of 4 to 40 °C, or in the range of 15 to 30 °C, or in the range of 18 to 25°C.
- the culture may be harvested, for example by centrifuging at 13000 g, for example at room temperature.
- the cell pellets may be resuspended in 150 ⁇ l of 1x lithium dodecyl sulphate (LDS) gel sample buffer and heated to the temperature, which may be in the range of 60 to 100 °C, or in the range of 80 to 98 °C, or in the range of 90 to 95 °C for the time period, which may be in the range of 1 to 60 minutes, or in the range of 2 to 30 minutes, or in the range of 3 to 10 minutes.
- LDS lithium dodecyl sulphate
- the samples may be spun down at 13000 g, for example at room temperature, and analyzed with for example gradient SDS-PAGE.
- the volume of the sample analyzed may be in the range of 1 ⁇ l to 15 ⁇ l, or in the range of 2 to 12 ⁇ l, or in the range of 5 to 10 ⁇ l.
- the purification of the FGF-2 polypeptide according to the invention may be carried out for example by the following procedure. Post-induction bacterial cells from one liter cultivation are harvested, for example by centrifugation at 5000 g for 20 minutes. The cell pellet is lysed, for example by addition of lysozyme and by sonication.
- the lysate is clarified by centrifugation, for example at 75000 g at 4 °C for 30 minutes, and filtered through a 0.22 ⁇ m membrane.
- the clarified lysate is loaded onto a HisTrap column.
- the FGF-2 polypeptide is eluted by imidazole and purified with cation exchange chromatography, for example using HiTrap SP Sepharose.
- the FGF-2 polypeptide may be analyzed by gel filtration chromatography, using for example Superdex200 Increase column (10/300).
- the thermostability of the FGF-2 polypeptide may be analyzed, for example by nanoDSF.
- the long-term stability of the FGF-2 polypeptides according to the invention may be performed.
- the FGF-2 polypeptide may be stored in a fridge, e.g. at 4 °C for 30 days, and the protein may be then analyzed by size-exclusion chromatography
- the stability analysis after one or more freeze-thaw cycles of the FGF-2 polypeptides according to the invention may be performed. For example by the process described in example 5.
- the biological activity of the FGF-2 polypeptides according to the invention may be tested and the achieved cell growth may be determined.
- the different concentrations of the FGF-2s may be tested, for example the concentration of FGF-2 may be 100, 10 or 1 ng/ml.
- the thermostabilized truncated FGF-2 (SEQ ID NO: 3) had a stronger biological effect than the commercial FGF-2.
- the melting temperature (Tm) was determined, and the effect of the mutations (deletion at the N-terminus and nine substitutions in the FGF-2 chain) to increase FGF-2 stability was evaluated.
- the FGF-2 polypeptides have been prepared in high yields.
- the products showed excellent thermal stability and very good long-term stability at 4°C.
- the products may be frozen and successfully recovered.
- the FGF-2 polypeptides according to the invention may be successfully used in biotechnological research and industrial applications, medicine, the pharmaceutical industry, cosmetics, clean meat industry, generation of organoids and 3D cell culture models and other related applications.
- the FGF-2 polypeptides according to the invention may be used in many biotechnological processes, for example for cell cultivation or for biotechnological production of many different types of requested compounds, such as for example proteins, active pharmaceutical ingredients or antibodies.
- the FGF-2 polypeptides according to the invention may be used in cell cultivation processes for the purpose of preparing the cultured meat products for human consumption or as a pet food.
- the FGF-2 polypeptides according to the invention may be used as a component of the culture media. These processes of cell cultivation may be carried out in a cell cultivation system 1, as depicted on Fig. 10.
- the cell cultivation system 1 may comprise at least one of: a cultivation device 2, formed for example by a production bioreactor, a cell harvesting device 3, a central control unit 4, or a monitoring device 5.
- the cultivation device 2, e.g. the production bioreactor, the cell harvesting device 3, the central control unit 4, and/or the monitoring device 5 may be in direct or indirect connection and/or communication with each other.
- the cell harvesting device 3 may comprise a filtration device, a centrifugation device, or any other appropriate device for harvesting of cells.
- the system may further comprise for example a seeding tank or a device for preparing cultured meat composition (not depicted on Fig.10).
- thermostable FGF-2 polypeptides according to the invention may be used as a component of the culture medium in may biotechnological processes, for example in cultivation of mammalian cells, for example for the purpose of preparing cultured meat products in clean meat industry.
- the thermostable FGF-2 polypeptides according to the invention may be used as signaling compounds in the culture medium.
- the culture medium according to the invention may further comprise amino acids or their sources, in combination with at least one type of compounds that may be selected from a group comprising: sugars, fatty acids, vitamins and organic micronutrients, mineral compounds, supplements, such as for example iron supplementation compounds, organic amines, shear protectants, additional compounds, or any other appropriate compounds.
- Example 1 preparation of the truncated thermostabilized FGF-2 polypeptide (SEQ ID NO: 3) The competent cells of three selected E. coli strains (BL21, BL-21-Gold and BL21-CodonPlus RIPL) were transformed with the plasmid DNA of the thermostabilized truncated construct FGF-2 corresponding to SEQ ID NO. 3, and each cell suspension was plated on agar plates supplemented with kanamycin. The next day, two colonies from each plate were picked into 5 mL LB broth supplemented with kanamycin and grown overnight.
- SEQ ID NO: 3 The competent cells of three selected E. coli strains (BL21, BL-21-Gold and BL21-CodonPlus RIPL) were transformed with the plasmid DNA of the thermostabilized truncated construct FGF-2 corresponding to SEQ ID NO. 3, and each cell suspension was plated on agar plates supplemented with kanamycin. The next day, two colonies from each plate were picked into 5 mL
- Fig.2 depicts the SDS-PAGE analysis of the expression of truncated thermostabilized FGF-2 characterized by SEQ ID NO: 3 in E. coli BL21 strain. The expression was induced by the addition of 1 mM IPTG and was performed at 20°C for 1, 3, 5, 8, 12, 18 and 24 h. The truncated thermostabilized FGF-2 (SEQ ID NO: 3) was successfully expressed in all three E.
- LDS lithium dodecyl sulphate
- Example 2 preparation of the truncated thermostabilized FGF-2 polypeptide (SEQ ID NO: 3) and the purification thereof on HisTrap FF column and Superdex200 Increase column
- the BL21 (DE3) competent cells were transformed with the plasmid DNA of truncated construct FGF-2 and plated on agar plates supplemented with kanamycin.
- the cells were harvested by centrifugation at 5000 g for 20 minutes.
- the cell pellet was resuspended in 100 mL of cold buffer containing 30 mM N-(2-Hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) - Hepes (pH 7.5), 500 mM NaCl, 10 mM imidazole, 10 mM MgCl2, 1% NP-40, 1 tablet of protease inhibitor SigmaFast, DNAse (5 mg in total), and lysozyme (100 mg in total). The suspension was incubated on ice for 30 minutes.
- the cells were lysed by sonication on ice for 8 min (10s On/20s Off, amplitude 40%), and the lysate was clarified by centrifugation at 75 000 g at 4 °C for 30 minutes.
- the lysate was filtered through a 0.22 ⁇ m membrane and loaded at a 5 mL/min flow rate onto the HisTrap FF (5 mL) column that was equilibrated in 30 mM HEPES (pH 7.5), 500 mM NaCl and 10 mM imidazole.
- the FGF-2 was washed with a buffer containing 30 mM HEPES (pH 7.5), 500 mM NaCl and 40 mM imidazole and finally eluted with 30 mM HEPES (pH 7.5), 500 mM NaCl and 150 mM imidazole.
- the protein was concentrated to 1 mL using VivaSpin Turbo centrifugal concentrators with 10 kDa cut-off and diluted 20 x with buffer containing 15 mM Hepes (pH 7.5).
- the diluted protein was loaded onto a HiTrap SP Sepharose HP (5 mL) at a flow rate of 5 mL/min and eluted with a continuous salt gradient of buffer containing 15 mM HEPES (pH 7.5) and 1 M NaCl.
- the total gradient elution time was 40 minutes, and the gradient was set from 0 to 60 % of the 15 mM HEPES (pH 7.5) and 1 M NaCl.
- the flow rate was 3 mL/min.
- the protein was concentrated to 0.5 mL using VivaSpin Turbo centrifugal concentrators with 10 kDa cut-off and loaded onto a Superdex200 Increase column (10/300).
- the isocratic elution from the Superdex200 Increase column was performed at 0.5 ml/min in a buffer containing 15 mM Hepes (pH 7.5) and 150 mM NaCl.
- the results of purification of the truncated FGF-2 characterized by SEQ ID NO: 3 were controlled using SDS PAGE analysis (Fig. 3). After sonication, the lysate was centrifuged, and 10 ⁇ l of supernatant (1) and pellet (2) were loaded on the gel.
- the supernatant was then loaded onto a HisTrap column and flow through (3), fraction after washing with buffer containing 10 mM imidazole (4), 40 mM imidazole (5), and finally with 150 mM imidazole (6) were loaded on the gel and analyzed using SDS PAGE.
- the elution profile of the truncated thermostabilized FGF-2 characterized by SEQ ID NO: 3 on the Superdex200 Increase 10/300 is shown in Fig. 4.
- the line with peak maximum at approximately 20.9 min represents conductivity.
- the upper line with peak maximum at approximately 17.6 min is absorbance at 280 nm
- the middle line is absorbance at 260 nm
- the bottom line is absorbance at 450 nm.
- thermostability assay The thermostability assay of FGF-2 was performed using nanoDSF in Prometheus NT.48 (NanoTemper). Standard NanoTemper capillaries were loaded with FGF-2 (1 mg/ml) in a buffer containing 15 mM Hepes (pH 7.5) and 150 mM NaCl. The measurement was performed in triplicates at temperatures from 20 °C to 90 °C with a temperature ramp of 1.5 °C/min. Changes in tryptophan emission at 330 and 350 nm were monitored, and the ratio 330/350 nm was plotted against the temperature.
- thermostabilized truncated FGF-2 corresponding to the SEQ ID NO: 3 determined by nanoDSF was 68.4 °C (Fig. 5). Representative thermal unfolding curve (top) and its first derivative analysis, indicating the melting temperature of the protein (bottom) are shown.
- the experimental Tm was about 14.9 °C higher than the Homo sapiens FGF-2 (SEQ ID NO: 1). This value indicates excellent thermal stability, which is in agreement with the contributions of individual substitutions.
- Example 4 long term stability
- the thermo-stabilized truncated FGF-2 corresponding to the SEQ ID NO: 3 was stored in a fridge at 4 °C for 30 days postproduction, and the protein was analyzed by size-exclusion chromatography in a buffer containing 15 mM Hepes (pH 7.5) and 150 mM NaCl in order to examine protein aggregation or degradation.
- the isocratic elution from the Superdex200 Increase column was performed at 0.5 ml/min in a buffer containing 15 mM Hepes (pH 7.5) and 150 mM NaCl.
- FIG. 6 depicts the elution profile of the truncated FGF-2 characterized by SEQ ID NO: 3 on Superdex200 Increase 10/300 after 30 days of storage at 4 °C.
- the bottom line is absorbance at 260 nm, the line above that is absorbance at 280 nm.
- the straight line above the absorbance at 280 nm is the system pressure, and the upper line shows conductivity.
- the thermostabilized truncated FGF-2 corresponding to the SEQ ID NO: 3 was eluted from Superdex200 Increase 10/300 column as the main peak.
- the elution volume was 19 mL and was comparable to the elution time observed on the first day of purification. No shift toward higher molecular weights was observed.
- Example 5 stability analysis after freeze-thaw cycle The stability analysis after one freeze-thaw cycle of the thermostabilized truncated FGF-2 characterized by SEQ ID NO: 3 was performed. The FGF-2 (1 mg/ml) in a buffer containing 15 mM Hepes (pH 7.5) and 150 mM NaCl was frozen in liquid nitrogen.
- Fig. 7 depicts the elution profile of the thermostabilized truncated FGF-2 corresponding to the SEQ ID NO: 3 on the Superdex200 Increase 10/300 column after freezing and thawing.
- the bottom line is the absorbance at 260 nm (lower main peak), the line above that is (higher main peak).
- the straight line above the absorbance at 280 nm is the system pressure and the upper line shows conductivity.
- FGF-2 eluted from the Superdex200 Increase 10/300 as the main peak.
- the elution volume was 19 mL and was comparable to the elution time observed on the first day of purification. No shift toward higher molecular weights was observed.
- a minor peak was observed at an elution volume of 21.1 mL and is probably a degradation product. Peak integration analysis indicated that the minor peak reflects 10% of the total FGF-2. About a 10% degradation of the thermostabilized truncated FGF-2 was observed after one freeze-thaw cycle.
- Example 6 testing the biological activity
- the biological effects of the FGF-2 polypeptide according to the invention have been tested and compared with commercial FGF-2 (Fig.8).
- Three different concentrations of the FGF-2s were tested: 100, 10 and 1 ng/ml.
- the achieved cell growth generally decreased with decreasing concentration of the FGF-2.
- the thermostabilized truncated FGF-2 SEQ ID NO: 3
- the achieved cell growth was comparable when concentrations of 100 and 10 ng/ml were applied, while a concentration of 1 ng/ml led to lower cell count.
- thermostabilized truncated FGF-2 (SEQ ID NO: 3) had a stronger biological effect than the commercial FGF-2. For example, 1.5 x, 2.8 x and 1.7 x higher cell count has been observed in the case of the 100 ng/ml, 10 ng/ml and 1 ng/ml concentrations of the prepared FGF-2 polypeptide, respectively.
- thermostabilized FGF-2 polypeptides according to the invention SEQ ID NO: 5 – 27
- the thermostabilized FGF-2 polypeptides according to the invention corresponding to SEQ ID NO: 5 – 27 have been prepared according to the procedure described in example 1.
- thermostabilized FGF-2 polypeptides depicts SDS-PAGE analysis of the expression of thermostabilized FGF-2 polypeptides.
- (1) non-induced cells (2) control - cells expressing the truncated thermostabilized FGF2 – SEQ ID NO: 3, (3) cells expressing dimeric thermostabilized FGF-2 - SEQ ID NO: 24, (4) cells expressing dimeric thermostabilized FGF-2 SEQ ID NO: 25, (5) cells expressing dimeric thermostabilized FGF-2 - SEQ ID NO: 26, (6) cells expressing dimeric thermostabilized FGF- 2 - SEQ ID NO: 27, (7) cells expressing thermostabilized FGF-2 - SEQ ID NO: 21, (8) cells expressing dimeric thermostabilized FGF-2 - SEQ ID NO: 22, (9) - cells expressing dimeric thermostabilized FGF-2 - SEQ ID NO: 23.
- the FGF-2 polypeptides with improved stability may be used in scientific research as well as in many industrial applications, for example, in biotechnological research, medicine, pharmaceutical industry, cosmetics, clean meat industry generation of organoids and 3D cell culture models and other related applications.
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Biochemistry (AREA)
- Gastroenterology & Hepatology (AREA)
- Zoology (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Toxicology (AREA)
- Peptides Or Proteins (AREA)
Abstract
of the Disclosure The invention relates to a Fibroblast Growth Factor 2 (FGF-2) polypeptides that have improved stability while maintaining biological activity compared to the wild-type of FGF-2. The invention provides the process for the preparation and the use thereof in biotechnological research and industrial applications, medicine, the pharmaceutical industry, cosmetics, clean meat industry, generation of organoids and 3D cell culture models and other related applications.
Description
THE FGF-2 POLYPEPTIDES WITH IMPROVED STABILITY, THE PROCESS FOR PREPARING THE FGF-2 POLYPEPTIDES AND USE THEREOF Field of the invention The invention relates to Fibroblast Growth Factor 2 (FGF-2) polypeptide with improved stability, especially thermal stability, compared to the wild-type of FGF-2 and the use thereof in research and industrial applications, such as biotechnological research, medicine, pharmaceutical industry, cosmetics, clean meat industry, generation of organoids and 3D cell culture models and other related applications. Background of the invention Fibroblast growth factors (FGFs) are a family of cell signaling proteins. They are involved in a variety of processes, especially as key elements for normal development in animal cells. These growth factors bind to heparin and heparan sulphate and typically activate cell surface receptors. Fibroblast growth factors that signal through FGF receptors (FGFRs) regulate fundamental developmental pathways, including the regulation of angiogenesis and wound repair. FGFRs are expressed on many different cell types and regulate key cell processes, such as proliferation, differentiation and survival, which make FGF signaling susceptible to subversion by cancer cells. FGFs are secreted glycoproteins that are generally readily sequestered to the extracellular matrix, as well as the cell surface, by heparan sulphate proteoglycans (HPSGs). For cellular signaling, FGFs are released from the extracellular matrix by heparinases, proteases or specific FGF-binding proteins, and the liberated FGFs subsequently bind to cell surface HPSGs. Cell surface HPSGs also stabilize the FGF ligand–receptor interaction, forming a ternary complex with FGFR. FGF receptors signal as dimers, and ligand-dependent dimerization leads to a conformational shift in receptor structure that activates the intracellular kinase domain, resulting in intermolecular transphosphorylation of the tyrosine kinase domains and intracellular tail. Phosphorylated tyrosine residues on the receptor function as docking sites for adaptor proteins, which themselves may also be directly phosphorylated by FGFR, leading to the activation of multiple signal transduction pathways. The human FGF-2 gene encodes not one protein, but a complex set of isoforms. The secreted isoform is a single-chain, non-glycosylated polypeptide with 154 amino acids. The amino acid sequence of human FGF-2 is 99% homologous to that of bovine FGF-2 and has high homology with ovine and rodent FGF-2, suggesting strong sequence conservation for structure and function. The stability of FGF-2 is widely accepted to be a major concern in the development of useful medicinal products or serum replacement in biotechnological research. The manufacturers usually state that solutions of reconstituted FGF-2 are stable for up to 12 months only when stored at -20 °C or lower. The reconstituted FGFs solutions are stable for only about a week
at 4 °C and are recommended to be used within 24 h when at ambient temperatures (around 25 °C). Nevertheless, a 50% loss of functionality of FGF-2 solutions at a concentration of 72 µg/mL was observed after just 4 min at 25 °C. The functional half-life is decreased to 37, 33 and 10 min, as the storage temperature is increased to 37 °C, 42 ◦C and 50 °C, respectively. One of the challenges is to preserve the biological activity of FGF-2. Most of these efforts were directed at sustaining FGF-2 activity for cell culture research, or to develop sustained-release formulations of FGF-2 for tissue engineering applications. For example, the approaches to maintain the stability and biological activity of FGF-2 include modulating ionic interactions in solution and chemical modifications of FGF-2. The addition of excipients to aqueous solutions of FGF-2 is among the simplest methods for FGF-2 stabilization by way of modulating ionic interactions in solution. Common strategies include the complexation of FGF-2 with its endogenous stabilizer, heparin or heparin-like polymers, or polycations. Ionic interactions between FGF-2 and the additives reduce the structural energy at the heparin-binding site, stabilize the FGF-2 native conformation and prolong its bioactivity in aqueous media. This method, unfortunately, has some disadvantages. For example, there are safety concerns because the pharmaceutical-grade heparins are isolated from porcine intestines and bovine lung tissues and the heparins are susceptible to batch-to-batch variability. This variability can induce immune responses or be contaminated or adulterated by natural and synthetic heparinoids that may lead to anaphylactoid type responses and death. Additionally, natural heparin is susceptible to degradation and desulphation by heparinases, which may adversely affect its effectiveness at stabilizing FGF-2. Methods of prolonging the bioactivity of FGF-2 in aqueous media include point mutations of the protein and covalent grafting of the protein onto scaffold materials. Mutant variants of the FGF-2 protein have been developed by aligning the wild-type FGF-2 protein sequence with stabilized FGF-1 mutant sequences, or by combining several individual stabilizing mutations identified by other investigators in the field. The identification of useful point mutations has been aided by computer modeling. Sequences have been identified that are important for protein functionality and contribute the greatest amount to structural free energy. Following an analysis of sequence conservation, mutations in regions, which might compromise protein function, were avoided. Conversely, mutations that were likely to result in a decrease in protein free energy were promoted. The obtained FGF-2 mutants showed the lowest energy and had increased functional half-life. Despite some advances within the state of the art, there is still need to further improve the stability of the FGF-2 product while maintaining its biological activity. The object of the invention is to prepare an FGF-2 variant with improved stability and to find an efficient, scalable and economically advantageous process for the production of FGF-2 with high yields. Brief summary of the invention The disadvantages of the solutions, according to the state of the art, are solved by the present invention that provides a thermostable polypeptide that possesses FGF-2 activity and has increased stability while maintaining biological activity compared to the wild-type FGF-2.
In one aspect of the invention, the product is truncated thermostabilized FGF-2 polypeptide with amino acid substitutions characterized by sequence SEQ ID NO: 3. According to the present invention, the FGF-2 polypeptide with SEQ ID NO: 3 has 80.6% sequence identity to Homo sapiens FGF-2 (SEQ ID NO: 1) and 81.3% sequence identity to Bos taurus FGF-2 (SEQ ID NO: 2). In another aspect of the invention, the product is a thermostabilized FGF-2 polypeptide with amino acid substitutions characterized by sequence SEQ ID NO: 4. The objects of the present invention are also other FGF-2 polypeptides that are derived from the amino acid sequence of SEQ ID NO: 3, for example FGF-2 polypeptides having at least 90% sequence identity, or at least 93% sequence identity, or at least 95% sequence identity to the sequence of the truncated thermostabilized FGF-2 polypeptide (SEQ ID NO: 3), FGF-2 polypeptides characterized by SEQ ID NO: 5-12 or SEQ ID NO:21 (which contains substitution L63Y), or other FGF-2 polypeptides according to the description below. The objects of the present invention are also other FGF-2 polypeptides that are derived from the amino acid sequence of SEQ ID NO: 4, for example FGF-2 polypeptides having at least 90% sequence identity, or at least 93% sequence identity, or at least 95% sequence identity to the sequence of the thermostabilized FGF-2 polypeptide (SEQ ID NO: 4), for example FGF-2 polypeptides characterized by SEQ ID NO: 13 to 20. The objects of the present invention are also dimeric variants of the FGF-2 polypeptide comprising a linker, for example GSS linker or SUMO linker. The objects of the present invention are also dimeric variants of the FGF-2 polypeptide characterized by SEQ ID NO: 22-27. The dimeric variants of the FGF-2 polypeptide may comprise two connected sequences characterized by SEQ ID NO: 3 or two connected sequences having at least 90%, or 93%, or 95% sequence identity to the SEQ ID NO: 3. The linker for connecting two sequences may be for example a GSS linker (e.g. 6xGSS or 10xGSS) as used in dimeric FGF-2 polypeptides characterized by SEQ ID NO: 22 – 23, or SUMO linkers characterized by SEQ ID NO: 28-31 as used in dimeric FGF-2 polypeptides characterized by SEQ ID NO: 24 - 27. The invention also provides the process for preparation of the FGF-2 polypeptides and the use thereof in the pharmaceutical industry, cosmetics, clean meat industry generation of organoids and 3D cell culture models and other related applications. The increased stability while maintaining biological activity compared to the Homo sapiens FGF-2 has been proven by experiments. The biological activity has been tested by cell growth experiments, the biological effects of commercially available FGF-2 and FGF-2 products according to the invention have been compared. According to the invention, the thermal stability of the FGF-2 polypeptides using nano differential scanning fluorimetry (nanoDSF) method, and the effect of long-term storage on product stability have been investigated.
Brief description of the several views of the drawings Fig. 1 – Sequence alignment of wild type FGF-2 homologs (Homo sapiens, Bos taurus) and FGF-2 products according to the invention characterized by SEQ ID NO: 3 and SEQ ID NO: 4 Fig. 2 – SDS-PAGE analysis of the expression of truncated thermostabilized FGF-2 characterized by SEQ ID NO: 3 in E. coli BL21 strain. The expression was induced by addition of 1 mM IPTG and was performed at 20°C for 1, 3, 5, 8, 12, 18 and 24 h Fig.3 – SDS-PAGE analysis of the truncated thermostabilized FGF-2 characterized by SEQ ID NO: 3 after purification on HisTrap column. (1) supernatant after sonication and centrifuging, (2) pellet after sonication and centrifuging, (3) flow through after loading the supernatant onto HisTrap column, (4) fraction after washing with buffer containing 10 mM imidazole, (5) fraction after washing with buffer containing 40 mM imidazole, (6) fraction after washing with buffer containing 150 mM imidazole Fig. 4 – Elution profile of the truncated thermostabilized FGF-2 characterized by SEQ ID NO: 3 on Superdex200 Increase 10/300. The line with peak maximum at approximately 20.9 min represents conductivity. The upper line with peak maximum at approximately 17.6 min is absorbance at 280 nm, the middle line is absorbance at 260 nm, and the bottom line is absorbance at 450 nm Fig.5 - Thermostability of truncated thermostabilized FGF-2 characterized by SEQ ID NO: 3 analyzed by nanoDSF. Changes in tryptophan emission at 330 and 350 nm were monitored, and the ratio 330/350 nm was plotted against the temperature. Representative thermal unfolding curve (top) and its first derivative analysis (bottom) Fig. 6 - Stability of truncated thermostabilized FGF-2 characterized by SEQ ID NO: 3 after storage at 4°C for 30 days Fig. 7 - Stability of truncated thermostabilized FGF-2 characterized by SEQ ID NO: 3 after freezing-thawing cycle Fig. 8 - The results from the cultivation of cells, when commercially available FGF-2 and truncated thermostabilized FGF-2 characterized by SEQ ID NO: 3 were compared Fig. 9 - SDS-PAGE analysis of the expression of thermostabilized FGF-2 polypeptides. (1) non-induced cells, (2) control - cells expressing the truncated thermostabilized FGF2 – SEQ ID NO: 3, (3) cells expressing dimeric thermostabilized FGF-2 - SEQ ID NO: 24, (4) cells expressing dimeric thermostabilized FGF-2 SEQ ID NO: 25, (5) cells expressing dimeric thermostabilized FGF-2 - SEQ ID NO: 26, (6) cells expressing dimeric thermostabilized FGF- 2 - SEQ ID NO: 27, (7) cells expressing thermostabilized FGF-2 - SEQ ID NO: 21, (8) cells expressing dimeric thermostabilized FGF-2 - SEQ ID NO: 22, (9) - cells expressing dimeric thermostabilized FGF-2 - SEQ ID NO: 23 Fig.10 - the cell cultivation system according to the invention
Detailed description of the invention The disadvantages of the solutions according to state of the art are solved by the present invention, that provides thermostable polypeptides, that possess FGF-2 activity and have increased stability compared to the wild-type FGF-2, while maintaining the biological activity. The invention also provides the process for preparation of the FGF-2 polypeptides and use thereof. In one aspect of the invention, the product is a thermostable FGF-2 polypeptide derived from Bos taurus FGF-2 (SEQ ID NO: 2) comprising at least one of the amino acids substitutions R31L, V52T, E54D, H59F, L92Y, S94I, C96N, S109E, S121P. In one aspect of the invention, the product is a truncated thermostabilized FGF-2 polypeptide characterized by sequence SEQ ID NO: 3, Fig. 1. The sequence SEQ ID NO: 3 is derived from the Bos taurus FGF-2 polypeptide (SEQ ID NO: 2), with the following modifications: deletion of amino acids 1-20 at the N-terminus, and nine amino acids substitutions, specifically R31L, V52T, E54D, H59F, L92Y, S94I, C96N, S109E, S121P. In comparison to Homo sapiens FGF-2 polypeptide (SEQ ID NO: 1), the Bos taurus FGF-2 (SEQ ID NO: 2) and also the truncated thermostabilized FGF-2 polypeptide (SEQ ID NO: 3) have the modification S137P. The nucleotide sequence encoding the truncated thermostabilized FGF-2 was cloned into the pET30a(+) expression vector. In other aspect of the invention, the FGF-2 polypeptides may be derived from the Bos taurus FGF-2 polypeptide (SEQ ID NO: 2), with deletion of amino acids 1-15 to 1-22 at the N- terminus. These polypeptides may comprise at least one of amino acids substitutions: R31L, V52T, E54D, H59F, L92Y, S94I, C96N, S109E, or S121P relative to SEQ ID NO: 2. The truncated thermostabilized FGF-2 polypeptide (SEQ ID NO: 3), according to the present invention, has 80.6% sequence identity to Homo sapiens FGF-2 (SEQ ID NO: 1) and 81.3% sequence identity to Bos taurus FGF-2 (SEQ ID NO: 2). In another aspect of the invention, the product is a thermostabilized FGF-2 polypeptide characterized by sequence SEQ ID NO: 4, Fig. 1. Nucleotide sequence encoding the thermostabilized FGF-2 was cloned into the pET30a(+) expression vector. The thermostabilized FGF-2 polypeptide (SEQ ID NO: 4), according to the present invention, has 93.5% sequence identity to Homo sapiens FGF-2 a sequence (SEQ ID NO: 1) and 94.2% sequence identity to Bos taurus FGF-2 (a sequence SEQ ID NO: 2). The objects of the present invention are also other FGF-2 polypeptides that are derived from the amino acid sequence of SEQ ID NO: 3, for example FGF-2 polypeptides having at least 90% sequence identity, or at least 93% sequence identity, or at least 95% sequence identity to the sequence of the truncated thermostabilized FGF-2 polypeptide (SEQ ID NO: 3). The objects of the present invention are also FGF-2 polypeptides derived from the amino acid sequence of SEQ ID NO: 3 comprising at least one of amino acids substitutions: R11L, V32T, E34D, H39F, L72Y, S74I, C76N, S89E, or S101P.
The objects of the present invention are also FGF-2 polypeptides characterized by SEQ ID NO: 5-12 or SEQ ID NO: 21 (which contains substitution L63Y), or other FGF-2 polypeptides according to the description below. The objects of the present invention are also other FGF-2 polypeptides that are derived from the amino acid sequence of SEQ ID NO: 4, for example FGF-2 polypeptides having at least 90% sequence identity, or at least 93% sequence identity, or at least 95% sequence identity to the sequence of the thermostabilized FGF-2 polypeptide (SEQ ID NO: 4), for example FGF- 2 polypeptides characterized by SEQ ID NO: 13 to 20. The thermostable FGF-2 polypeptide derived from the amino acid sequence of SEQ ID NO: 4 may comprise at least one of the amino acids substitutions R31L, V52T, E54D, H59F, L92Y, S94I, C96N, S109E, or S121P. In yet another aspect of the invention, the products are dimeric variants of the FGF-2 polypeptide comprising a linker, for example GSS linker or SUMO linker. The objects of the present invention are also dimeric variants of the FGF-2 polypeptide characterized by SEQ ID NO: 22-27. The dimeric variants of the FGF-2 polypeptide according to the invention may comprise two connected sequences characterized by SEQ ID NO: 3 or two connected sequences having at least 90%, or 93%, or 95% sequence identity to the SEQ ID NO: 3. The linker for connecting two sequences may be for example a GSS linker (6xGSS or 10xGSS) as used in dimeric FGF-2 polypeptides characterized by SEQ ID NO: 22 – 23, or SUMO linkers characterized by SEQ ID NO: 28-31 as used in dimeric FGF-2 polypeptides characterized by SEQ ID NO: 24 - 27. The thermostabilized FGF-2 polypeptide, according to the invention, may comprise amino acid substitution R31L (arginine at position 31 substituted by leucine) and H59F (histidine at position 59 substituted by phenylalanine) in the amino acid sequence of the Bos taurus FGF- 2 polypeptide (SEQ ID NO: 2). In yet another aspect of the invention, the thermo-stabilized FGF-2 polypeptide may comprise another amino acid substitutions than R31L and H59F substitution in SEQ ID NO: 2 or SEQ ID NO: 4. In one aspect of the invention, the thermostabilized FGF-2 polypeptide may comprise R31 substitution with any appropriate amino acid, for example with isoleucine or valine, and H59 substitution with any appropriate amino acid, for example with tryptophan or isoleucine. The object of the present invention is also FGF-2 polypeptides (SEQ ID NO: 5-12) that are derived from the amino acid sequence of SEQ ID NO: 3 and comprising the following two residues: (i) isoleucine, valine at position 11 of SEQ ID NO: 3 (i.e., R11I, R11V) (ii) tryptophan, isoleucine at position 39 of SEQ ID NO: 3 (i.e., H39W, H39I). The object of the present invention is also FGF-2 polypeptides (SEQ ID NO: 13-20) that are derived from the amino acid sequence of SEQ ID NO: 4 and comprising the following two residues: (i) isoleucine, valine at position 31 of SEQ ID NO: 4 (i.e., R31I, R31V) (ii) tryptophan, isoleucine at position 59 of SEQ ID NO: 4 (i.e., H59W, H59I).
The object of the present invention is also thermostabilized FGF-2 polypeptides with the melting temperature (Tm) higher than or equal to 55 °C, or higher than or equal to 65 °C, or higher than or equal to 68 °C. The melting temperature (Tm) of thermostabilized FGF-2 polypeptides according to the present invention may be in the range of 65 to 80 °C, or in the range of 67 to 75 °C, or in the range of 68 to 72 °C. The process according to the invention may consist of the steps: - Transformation of the competent E. coli cells with the plasmid DNA carrying the relevant FGF-2 sequence - Selection and screening of the transformed cells - Cultivation of the transformed cells - Induction of FGF-2 production - FGF-2 detection - FGF-2 isolation and purification - FGF-2 characterization. According to the process of the invention, the bacterial expression host system, for example E. coli bacterial expression host system, may be used for the production of FGF-2 polypeptides due to its low cost, well-known biochemistry and genetics, rapid growth, and good productivity. A bacterial expression system is ideal for FGF-2 production as FGF-2 is a relatively small and single-domain protein with a compact fold. Furthermore, crystal structures of FGF-FGFR-heparin complexes and molecular mechanisms of the action of FGF proteins suggest that no post-translational modifications or cofactors of FGF proteins are required for their proper function. Previously, FGF proteins purified from E. coli showed biological activity. Therefore, the bacterial expression host system appears to be ideal for the large-scale industrial production of FGF-2. The presence of rare codons may be addressed by using codon optimization. In one aspect of the invention, the strains used for the production of FGF-2 polypeptides according to the invention may be E. coli strains, for example, BL21, BL-21-Gold or BL21- CodonPlus RIPL. The competent cells for the production of FGF-2 polypeptides are transformed with the plasmid DNA carrying the relevant FGF-2 sequence, encoding for example the FGF- 2 polypeptide as defined by sequence SEQ ID NO: 3 or SEQ ID NO: 4, or any other sequences according to the invention, e.g. SEQ ID NO: 5 to 27. The transformed cells are plated onto solid selection media, for example suspension of cells may be plated on LB (Luria-Bertani) agar plates supplemented with antibiotics. The colonies are later, for example next day, picked and transferred into liquid selection media, for example LB broth supplemented with antibiotics. The cells are grown for several hours, for example overnight. The culture may be then placed into fresh liquid selection medium, for example LB broth supplemented with antibiotics, and incubated at temperature in the range of 15 to 45 °C, or in the range of 20 to 40 °C, or in the range of 30 to 37 °C. As an antibiotic may be used for example kanamycin, or any other appropriate antibiotic. When the optical density at 550 nm (OD550) in the range of 0.4 to 1.0, or in the range of 0.5 to 0.9, or in the range of 0.6 to 0.8 is reached, an inducing agent is added, for example isopropyl
β-D-thiogalactoside (IPTG), or any other appropriate inducing agent may be added to cell cultures for induction of FGF-2 production. The concentration of IPTG may be in the range of 0.001 to 10 mM, or in the range of 0.01 to 1 mM, or in the range of 0.05 to 0.5 mM. The cell cultures are incubated at a temperature in the range of 4 to 40 °C, or in the range of 15 to 30 °C, or in the range of 18 to 25°C. After the appropriate time of the incubation period, for example in the range 1 hour to 72 hours, or in the range of 2 hours to 48 hours, or in the range of 12 hours to 24 hours, the culture may be harvested, for example by centrifuging at 13000 g, for example at room temperature. The cell pellets may be resuspended in 150 µl of 1x lithium dodecyl sulphate (LDS) gel sample buffer and heated to the temperature, which may be in the range of 60 to 100 °C, or in the range of 80 to 98 °C, or in the range of 90 to 95 °C for the time period, which may be in the range of 1 to 60 minutes, or in the range of 2 to 30 minutes, or in the range of 3 to 10 minutes. The samples may be spun down at 13000 g, for example at room temperature, and analyzed with for example gradient SDS-PAGE. The volume of the sample analyzed may be in the range of 1 µl to 15 µl, or in the range of 2 to 12 µl, or in the range of 5 to 10 µl. The purification of the FGF-2 polypeptide according to the invention may be carried out for example by the following procedure. Post-induction bacterial cells from one liter cultivation are harvested, for example by centrifugation at 5000 g for 20 minutes. The cell pellet is lysed, for example by addition of lysozyme and by sonication. The lysate is clarified by centrifugation, for example at 75000 g at 4 °C for 30 minutes, and filtered through a 0.22 μm membrane. The clarified lysate is loaded onto a HisTrap column. The FGF-2 polypeptide is eluted by imidazole and purified with cation exchange chromatography, for example using HiTrap SP Sepharose. The FGF-2 polypeptide may be analyzed by gel filtration chromatography, using for example Superdex200 Increase column (10/300). The thermostability of the FGF-2 polypeptide may be analyzed, for example by nanoDSF. The long-term stability of the FGF-2 polypeptides according to the invention may be performed. For example the FGF-2 polypeptide may be stored in a fridge, e.g. at 4 °C for 30 days, and the protein may be then analyzed by size-exclusion chromatography The stability analysis after one or more freeze-thaw cycles of the FGF-2 polypeptides according to the invention may be performed. For example by the process described in example 5. The biological activity of the FGF-2 polypeptides according to the invention may be tested and the achieved cell growth may be determined. The different concentrations of the FGF-2s may be tested, for example the concentration of FGF-2 may be 100, 10 or 1 ng/ml. The thermostabilized truncated FGF-2 (SEQ ID NO: 3) had a stronger biological effect than the commercial FGF-2. For example, 1.5 x, 2.8 x and 1.7 x higher cell count has been observed in the case of the 100 ng/ml, 10 ng/ml and 1 ng/ml concentrations of the prepared FGF-2 polypeptide, respectively. Experimental conditions of testing biological activity may be as described in example 6, or any other appropriate conditions set according to the needs of a person skilled in the art.
The process for preparing the truncated thermostabilized FGF-2 and other FGF-2 derivatives, according to the invention, is appropriate for large-scale production. The process was verified for example in 1 liter scale and provided about 10 - 40 mg of FGF-2 with very high purity, for example, 94%. The melting temperature (Tm) was determined, and the effect of the mutations (deletion at the N-terminus and nine substitutions in the FGF-2 chain) to increase FGF-2 stability was evaluated. According to the invention, the FGF-2 polypeptides have been prepared in high yields. The products showed excellent thermal stability and very good long-term stability at 4°C. The products may be frozen and successfully recovered. The FGF-2 polypeptides according to the invention may be successfully used in biotechnological research and industrial applications, medicine, the pharmaceutical industry, cosmetics, clean meat industry, generation of organoids and 3D cell culture models and other related applications. These products according to the invention may be used for example for preparing the cosmetic products, such as creams, gels, lotions for improvement of a visual appearance of the skin and for skin rejuvenation. The FGF-2 polypeptides according to the invention may be used in many biotechnological processes, for example for cell cultivation or for biotechnological production of many different types of requested compounds, such as for example proteins, active pharmaceutical ingredients or antibodies. The FGF-2 polypeptides according to the invention may be used in cell cultivation processes for the purpose of preparing the cultured meat products for human consumption or as a pet food. The FGF-2 polypeptides according to the invention may be used as a component of the culture media. These processes of cell cultivation may be carried out in a cell cultivation system 1, as depicted on Fig. 10. The cell cultivation system 1 may comprise at least one of: a cultivation device 2, formed for example by a production bioreactor, a cell harvesting device 3, a central control unit 4, or a monitoring device 5. The cultivation device 2, e.g. the production bioreactor, the cell harvesting device 3, the central control unit 4, and/or the monitoring device 5 may be in direct or indirect connection and/or communication with each other. The cell harvesting device 3 may comprise a filtration device, a centrifugation device, or any other appropriate device for harvesting of cells. Optionally the system may further comprise for example a seeding tank or a device for preparing cultured meat composition (not depicted on Fig.10). The thermostable FGF-2 polypeptides according to the invention may be used as a component of the culture medium in may biotechnological processes, for example in cultivation of mammalian cells, for example for the purpose of preparing cultured meat products in clean meat industry. The thermostable FGF-2 polypeptides according to the invention may be used as signaling compounds in the culture medium. The culture medium according to the invention may further comprise amino acids or their sources, in combination with at least one type of compounds that may be selected from a group comprising: sugars, fatty acids, vitamins and organic micronutrients, mineral compounds, supplements, such as for example iron supplementation compounds, organic amines, shear protectants, additional compounds, or any other appropriate compounds.
EXAMPLES Example 1: preparation of the truncated thermostabilized FGF-2 polypeptide (SEQ ID NO: 3) The competent cells of three selected E. coli strains (BL21, BL-21-Gold and BL21-CodonPlus RIPL) were transformed with the plasmid DNA of the thermostabilized truncated construct FGF-2 corresponding to SEQ ID NO. 3, and each cell suspension was plated on agar plates supplemented with kanamycin. The next day, two colonies from each plate were picked into 5 mL LB broth supplemented with kanamycin and grown overnight. The following day, 50 µL of each overnight culture was added into two fresh 5 mL of LB broth supplemented with kanamycin, and incubated at 220 rpm at 37 °C. After 70 minutes, the cell cultures were let to cool down to 25 °C, which took 20 minutes. IPTG was added to a final concentration of 1 mM to induce the production of thermostabilized truncated FGF-2. Non-induced cell cultures were used as control samples. The cell cultures were incubated at 220 rpm at 25 °C for 24 hours. After the incubation period, 1 mL of each culture was pipetted into clean microcentrifuge tubes, and the cells were spun down at 13000 g at room temperature. The cell pellets were resuspended in 150 µL of 1x lithium dodecyl sulphate (LDS) gel sample buffer and heated to 95 °C for 5 minutes. The samples were spun down at 13000 g at room temperature, and 10 μl of each sample was loaded onto gradient SDS-PAGE. Fig.2 depicts the SDS-PAGE analysis of the expression of truncated thermostabilized FGF-2 characterized by SEQ ID NO: 3 in E. coli BL21 strain. The expression was induced by the addition of 1 mM IPTG and was performed at 20°C for 1, 3, 5, 8, 12, 18 and 24 h. The truncated thermostabilized FGF-2 (SEQ ID NO: 3) was successfully expressed in all three E. coli strains, BL21, BL21-Gold and BL21-CodonPlus RIPL. The FGF-2 protein was clearly visible between 15 and 20 kDa in cell cultures induced with IPTG. No band corresponding to truncated thermostabilized FGF-2 was observed in non-induced cells. The best yield was observed in BL21 (DE3) cells. Example 2: preparation of the truncated thermostabilized FGF-2 polypeptide (SEQ ID NO: 3) and the purification thereof on HisTrap FF column and Superdex200 Increase column The BL21 (DE3) competent cells were transformed with the plasmid DNA of truncated construct FGF-2 and plated on agar plates supplemented with kanamycin. The next day, acolony was picked and grew overnight in 5 mL LB broth supplemented with kanamycin. The following day, 1 mL of the overnight culture was added into fresh 1 L LB broth supplemented with kanamycin. Cultures (2x 0.5 L) in 2 L Erlenmeyer flask were incubated at 220 rpm at 37 °C. When the cell density reached OD550 of 0.6, the cultures were let to cool down on the ice to 25 °C, and this process took about 5 minutes. The final concentration of 1 mM IPTG was used to induce expression. After induction, the cells were grown at 220 rpm at 25°C. At 24 hours post-induction, the cells were harvested by centrifugation at 5000 g for 20 minutes. The cell pellet was resuspended in 100 mL of cold buffer containing 30 mM N-(2-Hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) - Hepes (pH 7.5), 500 mM NaCl, 10 mM imidazole, 10 mM MgCl2, 1% NP-40, 1 tablet of protease inhibitor SigmaFast, DNAse (5 mg in total), and lysozyme (100 mg in total). The suspension was incubated on ice for 30 minutes. Then, the cells were lysed by sonication on ice for 8 min (10s On/20s Off, amplitude 40%), and the lysate was clarified by centrifugation at 75 000 g at 4 °C for 30 minutes. The lysate was filtered through a 0.22 μm membrane and loaded at a 5 mL/min flow rate onto the HisTrap FF (5 mL) column that was equilibrated in 30 mM HEPES (pH 7.5), 500 mM NaCl and 10 mM imidazole. The FGF-2 was washed with a buffer containing 30 mM HEPES (pH
7.5), 500 mM NaCl and 40 mM imidazole and finally eluted with 30 mM HEPES (pH 7.5), 500 mM NaCl and 150 mM imidazole. The protein was concentrated to 1 mL using VivaSpin Turbo centrifugal concentrators with 10 kDa cut-off and diluted 20 x with buffer containing 15 mM Hepes (pH 7.5). The diluted protein was loaded onto a HiTrap SP Sepharose HP (5 mL) at a flow rate of 5 mL/min and eluted with a continuous salt gradient of buffer containing 15 mM HEPES (pH 7.5) and 1 M NaCl. The total gradient elution time was 40 minutes, and the gradient was set from 0 to 60 % of the 15 mM HEPES (pH 7.5) and 1 M NaCl. The flow rate was 3 mL/min. The protein was concentrated to 0.5 mL using VivaSpin Turbo centrifugal concentrators with 10 kDa cut-off and loaded onto a Superdex200 Increase column (10/300). The isocratic elution from the Superdex200 Increase column was performed at 0.5 ml/min in a buffer containing 15 mM Hepes (pH 7.5) and 150 mM NaCl. The results of purification of the truncated FGF-2 characterized by SEQ ID NO: 3 were controlled using SDS PAGE analysis (Fig. 3). After sonication, the lysate was centrifuged, and 10 µl of supernatant (1) and pellet (2) were loaded on the gel. The supernatant was then loaded onto a HisTrap column and flow through (3), fraction after washing with buffer containing 10 mM imidazole (4), 40 mM imidazole (5), and finally with 150 mM imidazole (6) were loaded on the gel and analyzed using SDS PAGE. The elution profile of the truncated thermostabilized FGF-2 characterized by SEQ ID NO: 3 on the Superdex200 Increase 10/300 is shown in Fig. 4. The line with peak maximum at approximately 20.9 min represents conductivity. The upper line with peak maximum at approximately 17.6 min is absorbance at 280 nm, the middle line is absorbance at 260 nm, and the bottom line is absorbance at 450 nm. Example 3: the thermostability assay The thermostability assay of FGF-2 was performed using nanoDSF in Prometheus NT.48 (NanoTemper). Standard NanoTemper capillaries were loaded with FGF-2 (1 mg/ml) in a buffer containing 15 mM Hepes (pH 7.5) and 150 mM NaCl. The measurement was performed in triplicates at temperatures from 20 °C to 90 °C with a temperature ramp of 1.5 °C/min. Changes in tryptophan emission at 330 and 350 nm were monitored, and the ratio 330/350 nm was plotted against the temperature. In one aspect of the invention, the Tm of thermostabilized truncated FGF-2 corresponding to the SEQ ID NO: 3 determined by nanoDSF was 68.4 °C (Fig. 5). Representative thermal unfolding curve (top) and its first derivative analysis, indicating the melting temperature of the protein (bottom) are shown. The experimental Tm was about 14.9 °C higher than the Homo sapiens FGF-2 (SEQ ID NO: 1). This value indicates excellent thermal stability, which is in agreement with the contributions of individual substitutions. Example 4: long term stability The thermo-stabilized truncated FGF-2 corresponding to the SEQ ID NO: 3 was stored in a fridge at 4 °C for 30 days postproduction, and the protein was analyzed by size-exclusion chromatography in a buffer containing 15 mM Hepes (pH 7.5) and 150 mM NaCl in order to examine protein aggregation or degradation. The isocratic elution from the Superdex200 Increase column was performed at 0.5 ml/min in a buffer containing 15 mM Hepes (pH 7.5) and 150 mM NaCl.
Fig. 6 depicts the elution profile of the truncated FGF-2 characterized by SEQ ID NO: 3 on Superdex200 Increase 10/300 after 30 days of storage at 4 °C. The bottom line is absorbance at 260 nm, the line above that is absorbance at 280 nm. The straight line above the absorbance at 280 nm is the system pressure, and the upper line shows conductivity. The thermostabilized truncated FGF-2 corresponding to the SEQ ID NO: 3 was eluted from Superdex200 Increase 10/300 column as the main peak. The elution volume was 19 mL and was comparable to the elution time observed on the first day of purification. No shift toward higher molecular weights was observed. Two minor peaks were detected at an elution volume of 20.1 mL and 21.1 mL and are probably products of degradation. Peak integration analysis indicates that the two minor peaks reflect 38% of the total FGF-2. The long-term stability analysis indicated that 38% of FGF-2 falls apart after 30 days of storage at 4°C. Example 5: stability analysis after freeze-thaw cycle The stability analysis after one freeze-thaw cycle of the thermostabilized truncated FGF-2 characterized by SEQ ID NO: 3 was performed. The FGF-2 (1 mg/ml) in a buffer containing 15 mM Hepes (pH 7.5) and 150 mM NaCl was frozen in liquid nitrogen. After one hour, the protein was thawed and loaded on a Superdex 200 Increase 10/300 column to examine protein aggregation or degradation. The isocratic elution from the Superdex200 Increase column was performed at 0.5 mL/min in a buffer containing 15 mM Hepes (pH 7.5) and 150 mM NaCl. Fig. 7 depicts the elution profile of the thermostabilized truncated FGF-2 corresponding to the SEQ ID NO: 3 on the Superdex200 Increase 10/300 column after freezing and thawing. The bottom line is the absorbance at 260 nm (lower main peak), the line above that is (higher main peak). The straight line above the absorbance at 280 nm is the system pressure and the upper line shows conductivity. FGF-2 eluted from the Superdex200 Increase 10/300 as the main peak. The elution volume was 19 mL and was comparable to the elution time observed on the first day of purification. No shift toward higher molecular weights was observed. A minor peak was observed at an elution volume of 21.1 mL and is probably a degradation product. Peak integration analysis indicated that the minor peak reflects 10% of the total FGF-2. About a 10% degradation of the thermostabilized truncated FGF-2 was observed after one freeze-thaw cycle. Example 6: testing the biological activity The biological effects of the FGF-2 polypeptide according to the invention (thermostabilized truncated FGF-2, SEQ ID NO: 3) have been tested and compared with commercial FGF-2 (Fig.8). Three different concentrations of the FGF-2s were tested: 100, 10 and 1 ng/ml. In the case of the commercial FGF-2, the achieved cell growth generally decreased with decreasing concentration of the FGF-2. In the case of the thermostabilized truncated FGF-2 (SEQ ID NO: 3), the achieved cell growth was comparable when concentrations of 100 and 10 ng/ml were applied, while a concentration of 1 ng/ml led to lower cell count.
The thermostabilized truncated FGF-2 (SEQ ID NO: 3) had a stronger biological effect than the commercial FGF-2. For example, 1.5 x, 2.8 x and 1.7 x higher cell count has been observed in the case of the 100 ng/ml, 10 ng/ml and 1 ng/ml concentrations of the prepared FGF-2 polypeptide, respectively. Experimental details of testing biological activity: ● Cells: C2C12 (seeding density 4.000 cells per well) ● Cultivation vessel: 24-well plate ● Basal medium: Essential 8™ Basal Medium ● FGF-2 variants tested: ○ No FGF-2 ○ Commercial FGF-2 - concentrations 1, 10 and 100 ng/ml Thermostabilized truncated FGF-2 (SEQ ID NO: 3) - concentrations 1, 10 and 100 ng/ml ● Cultivation time points: ○ Day 0 - seeding ○ Day 3 - cell counting ○ Day 7 - cell counting, end of experiment Example 7: preparation of other thermostabilized FGF-2 polypeptides according to the invention (SEQ ID NO: 5 – 27) The thermostabilized FGF-2 polypeptides according to the invention corresponding to SEQ ID NO: 5 – 27 have been prepared according to the procedure described in example 1. Fig.9 depicts SDS-PAGE analysis of the expression of thermostabilized FGF-2 polypeptides. (1) non-induced cells, (2) control - cells expressing the truncated thermostabilized FGF2 – SEQ ID NO: 3, (3) cells expressing dimeric thermostabilized FGF-2 - SEQ ID NO: 24, (4) cells expressing dimeric thermostabilized FGF-2 SEQ ID NO: 25, (5) cells expressing dimeric thermostabilized FGF-2 - SEQ ID NO: 26, (6) cells expressing dimeric thermostabilized FGF- 2 - SEQ ID NO: 27, (7) cells expressing thermostabilized FGF-2 - SEQ ID NO: 21, (8) cells expressing dimeric thermostabilized FGF-2 - SEQ ID NO: 22, (9) - cells expressing dimeric thermostabilized FGF-2 - SEQ ID NO: 23. Industrial Applicability According to the invention, the FGF-2 polypeptides with improved stability may be used in scientific research as well as in many industrial applications, for example, in biotechnological research, medicine, pharmaceutical industry, cosmetics, clean meat industry generation of organoids and 3D cell culture models and other related applications.
SEQUENCE LISTING: SEQ ID NO: 1 Homo sapiens FGF-2 MAAGSITTLPALPEDGGSGAFPPGHFKDPKRLYCKNGGFFLRIHPDGRVDGVREKSD PHIKLQLQAEERGVVSIKGVCANRYLAMKEDGRLLASKCVTDECFFFERLESNNYNT YRSRKYTSWYVALKRTGQYKLGSKTGPGQKAILFLPMSAKS SEQ ID NO: 2 Bos taurus FGF-2 MAAGSITTLPALPEDGGSGAFPPGHFKDPKRLYCKNGGFFLRIHPDGRVDGVREKSD PHIKLQLQAEERGVVSIKGVCANRYLAMKEDGRLLASKCVTDECFFFERLESNNYNT YRSRKYSSWYVALKRTGQYKLGPKTGPGQKAILFLPMSAKS SEQ ID NO: 3 truncated thermostabilized FGF-2 FPPGHFKDPKLLYCKNGGFFLRIHPDGRVDGTRDKSDPFIKLQLQAEERGVVSIKGVC ANRYLAMKEDGRLYAIKNVTDECFFFERLEENNYNTYRSRKYPSWYVALKRTGQY KLGPKTGPGQKAILFLPMSAKS SEQ ID NO: 4 thermostabilized FGF-2 MAAGSITTLPALPEDGGSGAFPPGHFKDPKLLYCKNGGFFLRIHPDGRVDGTRDKSD PFIKLQLQAEERGVVSIKGVCANRYLAMKEDGRLYAIKNVTDECFFFERLEENNYNT YRSRKYPSWYVALKRTGQYKLGPKTGPGQKAILFLPMSAKS SEQ ID NO: 5 FGF-2 polypeptide variant 5 FPPGHFKDPKILYCKNGGFFLRIHPDGRVDGTRDKSDPFIKLQLQAEERGVVSIKGVC ANRYLAMKEDGRLYAIKNVTDECFFFERLEENNYNTYRSRKYPSWYVALKRTGQY KLGPKTGPGQKAILFLPMSAKS SEQ ID NO: 6 FGF-2 polypeptide variant 6
FPPGHFKDPKVLYCKNGGFFLRIHPDGRVDGTRDKSDPFIKLQLQAEERGVVSIKGV CANRYLAMKEDGRLYAIKNVTDECFFFERLEENNYNTYRSRKYPSWYVALKRTGQ YKLGPKTGPGQKAILFLPMSAKS SEQ ID NO: 7 FGF-2 polypeptide variant 7 FPPGHFKDPKLLYCKNGGFFLRIHPDGRVDGTRDKSDPWIKLQLQAEERGVVSIKGV CANRYLAMKEDGRLYAIKNVTDECFFFERLEENNYNTYRSRKYPSWYVALKRTGQ YKLGPKTGPGQKAILFLPMSAKS SEQ ID NO: 8 FGF-2 polypeptide variant 8 FPPGHFKDPKLLYCKNGGFFLRIHPDGRVDGTRDKSDPIIKLQLQAEERGVVSIKGVC ANRYLAMKEDGRLYAIKNVTDECFFFERLEENNYNTYRSRKYPSWYVALKRTGQY KLGPKTGPGQKAILFLPMSAKS SEQ ID NO: 9 FGF-2 polypeptide variant 9 FPPGHFKDPKILYCKNGGFFLRIHPDGRVDGTRDKSDPWIKLQLQAEERGVVSIKGV CANRYLAMKEDGRLYAIKNVTDECFFFERLEENNYNTYRSRKYPSWYVALKRTGQ YKLGPKTGPGQKAILFLPMSAKS SEQ ID NO: 10 FGF-2 polypeptide variant 10 FPPGHFKDPKILYCKNGGFFLRIHPDGRVDGTRDKSDPIIKLQLQAEERGVVSIKGVC ANRYLAMKEDGRLYAIKNVTDECFFFERLEENNYNTYRSRKYPSWYVALKRTGQY KLGPKTGPGQKAILFLPMSAKS SEQ ID NO: 11 FGF-2 polypeptide variant 11 FPPGHFKDPKVLYCKNGGFFLRIHPDGRVDGTRDKSDPWIKLQLQAEERGVVSIKGV CANRYLAMKEDGRLYAIKNVTDECFFFERLEENNYNTYRSRKYPSWYVALKRTGQ YKLGPKTGPGQKAILFLPMSAKS
SEQ ID NO: 12 FGF-2 polypeptide variant 12 FPPGHFKDPKVLYCKNGGFFLRIHPDGRVDGTRDKSDPIIKLQLQAEERGVVSIKGVC ANRYLAMKEDGRLYAIKNVTDECFFFERLEENNYNTYRSRKYPSWYVALKRTGQY KLGPKTGPGQKAILFLPMSAKS SEQ ID NO: 13 FGF-2 polypeptide variant 13 MAAGSITTLPALPEDGGSGAFPPGHFKDPKILYCKNGGFFLRIHPDGRVDGTRDKSDP FIKLQLQAEERGVVSIKGVCANRYLAMKEDGRLYAIKNVTDECFFFERLEENNYNTY RSRKYPSWYVALKRTGQYKLGPKTGPGQKAILFLPMSAKS SEQ ID NO: 14 FGF-2 polypeptide variant 14 MAAGSITTLPALPEDGGSGAFPPGHFKDPKVLYCKNGGFFLRIHPDGRVDGTRDKSD PFIKLQLQAEERGVVSIKGVCANRYLAMKEDGRLYAIKNVTDECFFFERLEENNYNT YRSRKYPSWYVALKRTGQYKLGPKTGPGQKAILFLPMSAKS SEQ ID NO: 15 FGF-2 polypeptide variant 15 MAAGSITTLPALPEDGGSGAFPPGHFKDPKLLYCKNGGFFLRIHPDGRVDGTRDKSD PWIKLQLQAEERGVVSIKGVCANRYLAMKEDGRLYAIKNVTDECFFFERLEENNYN TYRSRKYPSWYVALKRTGQYKLGPKTGPGQKAILFLPMSAKS SEQ ID NO: 16 FGF-2 polypeptide variant 16 MAAGSITTLPALPEDGGSGAFPPGHFKDPKLLYCKNGGFFLRIHPDGRVDGTRDKSD PIIKLQLQAEERGVVSIKGVCANRYLAMKEDGRLYAIKNVTDECFFFERLEENNYNT YRSRKYPSWYVALKRTGQYKLGPKTGPGQKAILFLPMSAKS
SEQ ID NO: 17 FGF-2 polypeptide variant 17 MAAGSITTLPALPEDGGSGAFPPGHFKDPKILYCKNGGFFLRIHPDGRVDGTRDKSDP WIKLQLQAEERGVVSIKGVCANRYLAMKEDGRLYAIKNVTDECFFFERLEENNYNT YRSRKYPSWYVALKRTGQYKLGPKTGPGQKAILFLPMSAKS SEQ ID NO: 18 FGF-2 polypeptide variant 18 MAAGSITTLPALPEDGGSGAFPPGHFKDPKILYCKNGGFFLRIHPDGRVDGTRDKSDP IIKLQLQAEERGVVSIKGVCANRYLAMKEDGRLYAIKNVTDECFFFERLEENNYNTY RSRKYPSWYVALKRTGQYKLGPKTGPGQKAILFLPMSAKS SEQ ID NO: 19 FGF-2 polypeptide variant 19 MAAGSITTLPALPEDGGSGAFPPGHFKDPKVLYCKNGGFFLRIHPDGRVDGTRDKSD PWIKLQLQAEERGVVSIKGVCANRYLAMKEDGRLYAIKNVTDECFFFERLEENNYN TYRSRKYPSWYVALKRTGQYKLGPKTGPGQKAILFLPMSAKS SEQ ID NO: 20 FGF-2 polypeptide variant 20 MAAGSITTLPALPEDGGSGAFPPGHFKDPKVLYCKNGGFFLRIHPDGRVDGTRDKSD PIIKLQLQAEERGVVSIKGVCANRYLAMKEDGRLYAIKNVTDECFFFERLEENNYNT YRSRKYPSWYVALKRTGQYKLGPKTGPGQKAILFLPMSAKS SEQ ID NO: 21 FGF-2 polypeptide variant 21 FGF2_mut_trunc_L83Y FPPGHFKDPKLLYCKNGGFFLRIHPDGRVDGTRDKSDPFIKLQLQAEERGVVSIKGVC ANRYYAMKEDGRLYAIKNVTDECFFFERLEENNYNTYRSRKYPSWYVALKRTGQY KLGPKTGPGQKAILFLPMSAKS
SEQ ID NO: 22 FGF-2 dimeric polypeptide variant 22 FGF2_mut_trunc_GSS6x FPPGHFKDPKLLYCKNGGFFLRIHPDGRVDGTRDKSDPFIKLQLQAEERGVVSIKGVC ANRYLAMKEDGRLYAIKNVTDECFFFERLEENNYNTYRSRKYPSWYVALKRTGQY KLGPKTGPGQKAILFLPMSAKSGSSGSSGSSGSSGSSGSSFPPGHFKDPKLLYCKNGG FFLRIHPDGRVDGTRDKSDPFIKLQLQAEERGVVSIKGVCANRYLAMKEDGRLYAIK NVTDECFFFERLEENNYNTYRSRKYPSWYVALKRTGQYKLGPKTGPGQKAILFLPM SAKS SEQ ID NO: 23 FGF-2 dimeric polypeptide variant 23 FGF2_mut_trunc_GSS10x FPPGHFKDPKLLYCKNGGFFLRIHPDGRVDGTRDKSDPFIKLQLQAEERGVVSIKGVC ANRYLAMKEDGRLYAIKNVTDECFFFERLEENNYNTYRSRKYPSWYVALKRTGQY KLGPKTGPGQKAILFLPMSAKSGSSGSSGSSGSSGSSGSSGSSGSSGSSGSSFPPGHFK DPKLLYCKNGGFFLRIHPDGRVDGTRDKSDPFIKLQLQAEERGVVSIKGVCANRYLA MKEDGRLYAIKNVTDECFFFERLEENNYNTYRSRKYPSWYVALKRTGQYKLGPKTG PGQKAILFLPMSAKS SEQ ID NO: 24 FGF-2 dimeric polypeptide variant 24 FPPGHFKDPKLLYCKNGGFFLRIHPDGRVDGTRDKSDPFIKLQLQAEERGVVSIKGVC ANRYLAMKEDGRLYAIKNVTDECFFFERLEENNYNTYRSRKYPSWYVALKRTGQY KLGPKTGPGQKAILFLPMSAKSSITVRVRDQTGEETFFKIKKTTKMQKVFETYATRK GVQVNSLRFLLDGDRITPDQTPKMLELEDQDQIDCVLFPPGHFKDPKLLYCKNGGFF LRIHPDGRVDGTRDKSDPFIKLQLQAEERGVVSIKGVCANRYLAMKEDGRLYAIKNV TDECFFFERLEENNYNTYRSRKYPSWYVALKRTGQYKLGPKTGPGQKAILFLPMSA KS SEQ ID NO: 25 FGF-2 dimeric polypeptide variant 25 FPPGHFKDPKLLYCKNGGFFLRIHPDGRVDGTRDKSDPFIKLQLQAEERGVVSIKGVC ANRYLAMKEDGRLYAIKNVTDECFFFERLEENNYNTYRSRKYPSWYVALKRTGQY KLGPKTGPGQKAILFLPMSAKSGGGGSSSITVRVRDQTGEETFFKIKKTTKMQKVFET YATRKGVQVNSLRFLLDGDRITPDQTPKMLELEDQDQIDCVLGGGGSSFPPGHFKDP
KLLYCKNGGFFLRIHPDGRVDGTRDKSDPFIKLQLQAEERGVVSIKGVCANRYLAM KEDGRLYAIKNVTDECFFFERLEENNYNTYRSRKYPSWYVALKRTGQYKLGPKTGP GQKAILFLPMSAKS SEQ ID NO: 26 FGF-2 dimeric polypeptide variant 26 FPPGHFKDPKLLYCKNGGFFLRIHPDGRVDGTRDKSDPFIKLQLQAEERGVVSIKGVC ANRYLAMKEDGRLYAIKNVTDECFFFERLEENNYNTYRSRKYPSWYVALKRTGQY KLGPKTGPGQKAILFLPMSAKSPSPSPSSITVRVRDQTGEETFFKIKKTTKMQKVFETY ATRKGVQVNSLRFLLDGDRITPDQTPKMLELEDQDQIDCVLPSPSPSFPPGHFKDPKL LYCKNGGFFLRIHPDGRVDGTRDKSDPFIKLQLQAEERGVVSIKGVCANRYLAMKE DGRLYAIKNVTDECFFFERLEENNYNTYRSRKYPSWYVALKRTGQYKLGPKTGPGQ KAILFLPMSAKS SEQ ID NO: 27 FGF-2 dimeric polypeptide variant 27 FPPGHFKDPKLLYCKNGGFFLRIHPDGRVDGTRDKSDPFIKLQLQAEERGVVSIKGVC ANRYLAMKEDGRLYAIKNVTDECFFFERLEENNYNTYRSRKYPSWYVALKRTGQY KLGPKTGPGQKAILFLPMSAKSMDVPATTEDVKESAESITVRVRDQTGEETFFKIKKT TKMQKVFETYATRKGVQVNSLRFLLDGDRITPDQTPKMLELEDQDQIDCVLEQTGG KGHGQIGAAAQFPPGHFKDPKLLYCKNGGFFLRIHPDGRVDGTRDKSDPFIKLQLQA EERGVVSIKGVCANRYLAMKEDGRLYAIKNVTDECFFFERLEENNYNTYRSRKYPS WYVALKRTGQYKLGPKTGPGQKAILFLPMSAKS SEQ ID NO: 28 SUMO short SITVRVRDQTGEETFFKIKKTTKMQKVFETYATRKGVQVNSLRFLLDGDRITPDQTP KMLELEDQDQIDCVL SEQ ID NO: 29 SUMO short fl GGGGSSSITVRVRDQTGEETFFKIKKTTKMQKVFETYATRKGVQVNSLRFLLDGDRI TPDQTPKMLELEDQDQIDCVLGGGGSS
SEQ ID NO: 30 SUMO short rl PSPSPSSITVRVRDQTGEETFFKIKKTTKMQKVFETYATRKGVQVNSLRFLLDGDRIT PDQTPKMLELEDQDQIDCVLPSPSPS SEQ ID NO: 31 SUMO long MDVPATTEDVKESAESITVRVRDQTGEETFFKIKKTTKMQKVFETYATRKGVQVNS LRFLLDGDRITPDQTPKMLELEDQDQIDCVLEQTGGKGHGQIGAAAQ
Claims
Claims 1. A thermostable FGF-2 polypeptide derived from Bos taurus FGF-2 (SEQ ID NO: 2) comprising at least one of the amino acids substitutions: R31L, V52T, E54D, H59F, L92Y, S94I, C96N, S109E, or S121P.
2. A thermostable FGF-2 polypeptide derived from the Bos taurus FGF-2 polypeptide (SEQ ID NO: 2) characterized by deletion of amino acids 1-15 to 1-22 at the N-terminus.
3. The thermostable FGF-2 polypeptide according to claim 2 derived from the Bos taurus FGF- 2 polypeptide (SEQ ID NO: 2) characterized by deletion of amino acids 1-20 at the N-terminus.
4. The thermostable FGF-2 polypeptide according to claim 3 comprising at least one of amino acids substitutions: R11L, V32T, E34D, H39F, L72Y, S74I, C76N, S89E, or S101P.
5. A thermostable FGF-2 polypeptide having at least 90% sequence identity to SEQ ID NO: 3.
6. The thermostable FGF-2 polypeptide according to claim 5 having at least 93% sequence identity to SEQ ID NO: 3.
7. The thermostable FGF-2 polypeptide according to claim 5 comprising at least one amino acid of isoleucine or valine at position 11 of SEQ ID NO: 3.
8. The thermostable FGF-2 polypeptide according to claim 5 comprising at least one amino acid of tryptophan or isoleucine at position 39 of SEQ ID NO: 3.
9. The thermostable FGF-2 polypeptide according to claim 5 characterized by SEQ ID NO: 3.
10. A thermostable FGF-2 polypeptide having at least 90 % sequence identity to SEQ ID NO: 4.
11. The thermostable FGF-2 polypeptide according to claim 10 comprising at least one amino acid of isoleucine or valine at position 31 of SEQ ID NO: 4.
12. The thermostable FGF-2 polypeptide according to claim 10 comprising at least one amino acid of tryptophan or isoleucine at position 59 of SEQ ID NO: 4.
13. The thermostable FGF-2 polypeptide according to claim 10 comprising at least one of the amino acids substitutions: R31L, V52T, E54D, H59F, L92Y, S94I, C96N, S109E, or S121P.
14. The thermostable FGF-2 polypeptide according to claim 10 characterized by SEQ ID NO: 4.
15. The thermostable FGF-2 polypeptide according to claim 1, characterized by sequences SEQ ID NO: 5-21.
16. A dimeric thermostable FGF-2 polypeptide comprising GSS linker or SUMO linker. 1
17. A dimeric FGF-2 polypeptide according to the claim 16 comprising at least one sequence from sequences SEQ ID NO: 1 to SEQ ID NO: 21 and a linker.
18. The dimeric FGF-2 polypeptide according to claim 17 comprising the SEQ ID NO: 3.
19. A dimeric FGF-2 polypeptide characterized by sequences SEQ ID NO: 22 to SEQ ID NO: 27.
20. A thermostable FGF-2 polypeptide having the melting temperature (Tm) higher than or equal to 55 °C.
21. The thermostable FGF-2 polypeptide according to claim 20 having the melting temperature (Tm) higher than or equal to 65 °C.
22. The thermostable FGF-2 polypeptide according to claim 20 having the melting temperature (Tm) higher than or equal to 68 °C.
23. Use of thermostable FGF-2 polypeptide according to any preceeding claims in preparing cultured meat products.
24. Use of thermostable FGF-2 polypeptide according to claim 23 in preparing cultured meat products, wherein the process of cell cultivation is carried out in a cell cultivation system 1 comprising at least one of: a cell cultivation device 2, a cell harvesting device 3, a central control unit 4, or a monitoring device 5.
25. Use of thermostable FGF-2 polypeptide according to claims 1 to 22 in cosmetics.
26. A culture medium comprising thermostable FGF-2 polypeptide according to claims 1 to 22.
27. The culture medium according to claim 26 comprising thermostable FGF-2 polypeptide characterized by SEQ ID NO: 3 to 27. 2
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US202263384999P | 2022-11-25 | 2022-11-25 | |
| US63/384,999 | 2022-11-25 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO2024110932A1 true WO2024110932A1 (en) | 2024-05-30 |
Family
ID=89068774
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/IB2023/061877 WO2024110932A1 (en) | 2022-11-25 | 2023-11-24 | The fgf-2 polypeptides with improved stability, the process for preparing the fgf-2 polypeptides and use thereof |
Country Status (1)
| Country | Link |
|---|---|
| WO (1) | WO2024110932A1 (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2013006544A1 (en) * | 2011-07-06 | 2013-01-10 | Medimmune, Llc | Methods for making multimeric polypeptides |
| WO2013090919A1 (en) * | 2011-12-16 | 2013-06-20 | Wisconsin Alumni Research Foundation | Fgf-2 having enhanced stability |
| WO2017089016A1 (en) * | 2015-11-27 | 2017-06-01 | Masarykova Univerzita | Thermostable fgf2 polypeptide, use thereof |
| US20200188267A1 (en) * | 2015-06-04 | 2020-06-18 | PnP Biopharm Co., Ltd. | Human fibroblast growth factor-2 mutant with increased stability, and use thereof |
| US20220025334A1 (en) * | 2018-12-12 | 2022-01-27 | Wild Type, Inc. | Synthetic food compositions |
-
2023
- 2023-11-24 WO PCT/IB2023/061877 patent/WO2024110932A1/en unknown
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2013006544A1 (en) * | 2011-07-06 | 2013-01-10 | Medimmune, Llc | Methods for making multimeric polypeptides |
| WO2013090919A1 (en) * | 2011-12-16 | 2013-06-20 | Wisconsin Alumni Research Foundation | Fgf-2 having enhanced stability |
| US20200188267A1 (en) * | 2015-06-04 | 2020-06-18 | PnP Biopharm Co., Ltd. | Human fibroblast growth factor-2 mutant with increased stability, and use thereof |
| WO2017089016A1 (en) * | 2015-11-27 | 2017-06-01 | Masarykova Univerzita | Thermostable fgf2 polypeptide, use thereof |
| US20220025334A1 (en) * | 2018-12-12 | 2022-01-27 | Wild Type, Inc. | Synthetic food compositions |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US11396537B2 (en) | Polypeptide, process for the production thereof and use thereof | |
| US5453490A (en) | Production of recombinant human interleukin-1 inhibitor | |
| US11746135B2 (en) | Thermostable FGF2 polypeptide, use thereof | |
| Yoshizumi et al. | Self‐association of streptococcus pyogenes collagen‐like constructs into higher order structures | |
| KR101778202B1 (en) | A human basic fibroblast factor-2 mutant with high stability and use of the same | |
| AU2016359722A1 (en) | Thermostable FGF2 polypeptide, use thereof | |
| KR20160064192A (en) | Protoxin-ii variants and methods of use | |
| Guo et al. | Production of soluble bioactive mouse leukemia inhibitory factor from Escherichia coli using MBP tag | |
| PT98711B (en) | METHOD FOR THE PREPARATION OF ALAGLU-IGF-1 AND ITS RENATURATION AND METHOD FOR THE PREPARATION OF HUMAN IGF-1 | |
| Gasparian et al. | Overexpression in Escherichia coli and purification of human fibroblast growth factor (FGF-2) | |
| WO2024110932A1 (en) | The fgf-2 polypeptides with improved stability, the process for preparing the fgf-2 polypeptides and use thereof | |
| JP3395181B2 (en) | Hematopoietic stem cell augmentation agent | |
| CN105176908A (en) | Producing method of recombination human fibroblast growth factor (FGF)-18 | |
| KR101754272B1 (en) | A human basic fibroblast factor-2 mutant with high stability and use of the same | |
| Song et al. | High-efficiency production of bioactive recombinant human fibroblast growth factor 18 in Escherichia coli and its effects on hair follicle growth | |
| Buchner et al. | [32] Purification and characterization of prokaryotic and eukaryotic Hsp90 | |
| WO2007080847A1 (en) | Mutant growth factor with altered receptor specificity and pharmaceutical composition containing the same | |
| KR102084065B1 (en) | Thermostable recombinant cellulase b protein derived from thermotoga maritima and the uses thereof | |
| KR20160083383A (en) | High-Stable Mutant of Basic Fibroblast Growth Factor, And Uses Thereof | |
| JP2611102B2 (en) | Method for producing biologically active β-NGF by genetic manipulation | |
| Bullock et al. | Expression and production of pigment epithelium-derived factor (PEDF) and PEDF receptor variants from mammalian and bacterial cells | |
| CN117903287A (en) | Recombinant type IV collagen with antioxidant activity and application thereof | |
| Wu et al. | Cost-Effective Expression of Human Bio-Identical Basic Fibroblast Growth Factor in Bacillus subtilis by Employing Asp DnaE Protein Intron | |
| Roussel et al. | ACCELERATED COMMUNICATION | |
| Liu et al. | Computer-assisted engineering of stable human leukemia inhibitory factor |