CN102199214B - Protein preparation method - Google Patents
Protein preparation method Download PDFInfo
- Publication number
- CN102199214B CN102199214B CN 201110062686 CN201110062686A CN102199214B CN 102199214 B CN102199214 B CN 102199214B CN 201110062686 CN201110062686 CN 201110062686 CN 201110062686 A CN201110062686 A CN 201110062686A CN 102199214 B CN102199214 B CN 102199214B
- Authority
- CN
- China
- Prior art keywords
- peptide
- hydrazides
- solution
- polypeptide
- tyr
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 238000003257 protein preparation method Methods 0.000 title abstract 3
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 201
- 229920001184 polypeptide Polymers 0.000 claims abstract description 121
- 102000004196 processed proteins & peptides Human genes 0.000 claims abstract description 121
- 238000000034 method Methods 0.000 claims abstract description 86
- -1 thioester compounds Chemical class 0.000 claims abstract description 41
- 230000007935 neutral effect Effects 0.000 claims abstract description 39
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 29
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 29
- IVRMZWNICZWHMI-UHFFFAOYSA-N azide group Chemical group [N-]=[N+]=[N-] IVRMZWNICZWHMI-UHFFFAOYSA-N 0.000 claims abstract description 17
- 150000007970 thio esters Chemical group 0.000 claims abstract description 14
- 239000011541 reaction mixture Substances 0.000 claims abstract description 8
- 125000003396 thiol group Chemical group [H]S* 0.000 claims abstract description 7
- 229940042795 hydrazides for tuberculosis treatment Drugs 0.000 claims description 123
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 87
- 239000002253 acid Substances 0.000 claims description 32
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 claims description 31
- 239000002994 raw material Substances 0.000 claims description 22
- 239000003795 chemical substances by application Substances 0.000 claims description 18
- 230000002829 reductive effect Effects 0.000 claims description 18
- 238000002360 preparation method Methods 0.000 claims description 13
- 239000000203 mixture Substances 0.000 claims description 12
- ORXSLDYRYTVAPC-UHFFFAOYSA-N 2-(4-sulfanylphenyl)acetic acid Chemical group OC(=O)CC1=CC=C(S)C=C1 ORXSLDYRYTVAPC-UHFFFAOYSA-N 0.000 claims description 6
- 230000015572 biosynthetic process Effects 0.000 claims description 4
- 239000000243 solution Substances 0.000 abstract description 128
- 238000006243 chemical reaction Methods 0.000 abstract description 54
- 239000011259 mixed solution Substances 0.000 abstract description 4
- 125000002252 acyl group Chemical group 0.000 abstract description 3
- 230000002378 acidificating effect Effects 0.000 abstract description 2
- 238000002156 mixing Methods 0.000 abstract description 2
- 239000003638 chemical reducing agent Substances 0.000 abstract 2
- 235000018102 proteins Nutrition 0.000 abstract 2
- LPXPTNMVRIOKMN-UHFFFAOYSA-M sodium nitrite Chemical compound [Na+].[O-]N=O LPXPTNMVRIOKMN-UHFFFAOYSA-M 0.000 abstract 2
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 abstract 1
- 235000018417 cysteine Nutrition 0.000 abstract 1
- 235000010288 sodium nitrite Nutrition 0.000 abstract 1
- PJJJBBJSCAKJQF-UHFFFAOYSA-N guanidinium chloride Chemical compound [Cl-].NC(N)=[NH2+] PJJJBBJSCAKJQF-UHFFFAOYSA-N 0.000 description 69
- 239000000047 product Substances 0.000 description 47
- KXDAEFPNCMNJSK-UHFFFAOYSA-N Benzamide Chemical compound NC(=O)C1=CC=CC=C1 KXDAEFPNCMNJSK-UHFFFAOYSA-N 0.000 description 46
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 34
- 238000004128 high performance liquid chromatography Methods 0.000 description 28
- 238000003756 stirring Methods 0.000 description 25
- 239000007864 aqueous solution Substances 0.000 description 24
- 239000011521 glass Substances 0.000 description 24
- 238000001819 mass spectrum Methods 0.000 description 24
- PZBFGYYEXUXCOF-UHFFFAOYSA-N TCEP Chemical compound OC(=O)CCP(CCC(O)=O)CCC(O)=O PZBFGYYEXUXCOF-UHFFFAOYSA-N 0.000 description 23
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 23
- 239000012295 chemical reaction liquid Substances 0.000 description 23
- 230000001143 conditioned effect Effects 0.000 description 23
- 229960000789 guanidine hydrochloride Drugs 0.000 description 23
- 230000003647 oxidation Effects 0.000 description 23
- 238000007254 oxidation reaction Methods 0.000 description 23
- NBIIXXVUZAFLBC-UHFFFAOYSA-N phosphoric acid Substances OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 23
- 150000003016 phosphoric acids Chemical class 0.000 description 23
- 239000011347 resin Substances 0.000 description 21
- 229920005989 resin Polymers 0.000 description 21
- 238000005516 engineering process Methods 0.000 description 13
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 9
- 230000004927 fusion Effects 0.000 description 8
- 101000654319 Centruroides suffusus Beta-mammal toxin Css2 Proteins 0.000 description 7
- 150000001413 amino acids Chemical class 0.000 description 7
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 description 6
- 238000005406 washing Methods 0.000 description 6
- VHYFNPMBLIVWCW-UHFFFAOYSA-N 4-Dimethylaminopyridine Chemical compound CN(C)C1=CC=NC=C1 VHYFNPMBLIVWCW-UHFFFAOYSA-N 0.000 description 5
- 239000003875 Wang resin Substances 0.000 description 5
- NERFNHBZJXXFGY-UHFFFAOYSA-N [4-[(4-methylphenyl)methoxy]phenyl]methanol Chemical compound C1=CC(C)=CC=C1COC1=CC=C(CO)C=C1 NERFNHBZJXXFGY-UHFFFAOYSA-N 0.000 description 5
- 241000894006 Bacteria Species 0.000 description 4
- 108020004414 DNA Proteins 0.000 description 4
- 210000004027 cell Anatomy 0.000 description 4
- 150000001875 compounds Chemical class 0.000 description 4
- 238000005520 cutting process Methods 0.000 description 4
- 238000006698 hydrazinolysis reaction Methods 0.000 description 4
- NPZTUJOABDZTLV-UHFFFAOYSA-N hydroxybenzotriazole Substances O=C1C=CC=C2NNN=C12 NPZTUJOABDZTLV-UHFFFAOYSA-N 0.000 description 4
- 239000006166 lysate Substances 0.000 description 4
- 230000004048 modification Effects 0.000 description 4
- 238000012986 modification Methods 0.000 description 4
- 239000007790 solid phase Substances 0.000 description 4
- 238000010532 solid phase synthesis reaction Methods 0.000 description 4
- 108010062785 trifolitoxin Proteins 0.000 description 4
- WFDIJRYMOXRFFG-UHFFFAOYSA-N Acetic anhydride Chemical compound CC(=O)OC(C)=O WFDIJRYMOXRFFG-UHFFFAOYSA-N 0.000 description 3
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 3
- SJRJJKPEHAURKC-UHFFFAOYSA-N N-Methylmorpholine Chemical compound CN1CCOCC1 SJRJJKPEHAURKC-UHFFFAOYSA-N 0.000 description 3
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 238000013467 fragmentation Methods 0.000 description 3
- 238000006062 fragmentation reaction Methods 0.000 description 3
- 230000006870 function Effects 0.000 description 3
- 230000000968 intestinal effect Effects 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 150000003053 piperidines Chemical class 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 230000003161 proteinsynthetic effect Effects 0.000 description 3
- 238000010189 synthetic method Methods 0.000 description 3
- NWZSZGALRFJKBT-KNIFDHDWSA-N (2s)-2,6-diaminohexanoic acid;(2s)-2-hydroxybutanedioic acid Chemical compound OC(=O)[C@@H](O)CC(O)=O.NCCCC[C@H](N)C(O)=O NWZSZGALRFJKBT-KNIFDHDWSA-N 0.000 description 2
- 125000003088 (fluoren-9-ylmethoxy)carbonyl group Chemical group 0.000 description 2
- 229960000549 4-dimethylaminophenol Drugs 0.000 description 2
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 241000713772 Human immunodeficiency virus 1 Species 0.000 description 2
- 102000035195 Peptidases Human genes 0.000 description 2
- 108091005804 Peptidases Proteins 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 150000001540 azides Chemical class 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 230000000975 bioactive effect Effects 0.000 description 2
- 230000001851 biosynthetic effect Effects 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000002330 electrospray ionisation mass spectrometry Methods 0.000 description 2
- IKDUDTNKRLTJSI-UHFFFAOYSA-N hydrazine monohydrate Substances O.NN IKDUDTNKRLTJSI-UHFFFAOYSA-N 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 230000017730 intein-mediated protein splicing Effects 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 239000000178 monomer Substances 0.000 description 2
- 108010005636 polypeptide C Proteins 0.000 description 2
- 238000001243 protein synthesis Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 230000008961 swelling Effects 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- 230000014616 translation Effects 0.000 description 2
- DTQVDTLACAAQTR-UHFFFAOYSA-N trifluoroacetic acid Substances OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 2
- HBGPNLPABVUVKZ-POTXQNELSA-N (1r,3as,4s,5ar,5br,7r,7ar,11ar,11br,13as,13br)-4,7-dihydroxy-3a,5a,5b,8,8,11a-hexamethyl-1-prop-1-en-2-yl-2,3,4,5,6,7,7a,10,11,11b,12,13,13a,13b-tetradecahydro-1h-cyclopenta[a]chrysen-9-one Chemical compound C([C@@]12C)CC(=O)C(C)(C)[C@@H]1[C@H](O)C[C@]([C@]1(C)C[C@@H]3O)(C)[C@@H]2CC[C@H]1[C@@H]1[C@]3(C)CC[C@H]1C(=C)C HBGPNLPABVUVKZ-POTXQNELSA-N 0.000 description 1
- VYMPLPIFKRHAAC-UHFFFAOYSA-N 1,2-ethanedithiol Chemical compound SCCS VYMPLPIFKRHAAC-UHFFFAOYSA-N 0.000 description 1
- PFRGGOIBYLYVKM-UHFFFAOYSA-N 15alpha-hydroxylup-20(29)-en-3-one Natural products CC(=C)C1CCC2(C)CC(O)C3(C)C(CCC4C5(C)CCC(=O)C(C)(C)C5CCC34C)C12 PFRGGOIBYLYVKM-UHFFFAOYSA-N 0.000 description 1
- 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 1
- 108010053481 Antifreeze Proteins Proteins 0.000 description 1
- OBMZMSLWNNWEJA-XNCRXQDQSA-N C1=CC=2C(C[C@@H]3NC(=O)[C@@H](NC(=O)[C@H](NC(=O)N(CC#CCN(CCCC[C@H](NC(=O)[C@@H](CC4=CC=CC=C4)NC3=O)C(=O)N)CC=C)NC(=O)[C@@H](N)C)CC3=CNC4=C3C=CC=C4)C)=CNC=2C=C1 Chemical compound C1=CC=2C(C[C@@H]3NC(=O)[C@@H](NC(=O)[C@H](NC(=O)N(CC#CCN(CCCC[C@H](NC(=O)[C@@H](CC4=CC=CC=C4)NC3=O)C(=O)N)CC=C)NC(=O)[C@@H](N)C)CC3=CNC4=C3C=CC=C4)C)=CNC=2C=C1 OBMZMSLWNNWEJA-XNCRXQDQSA-N 0.000 description 1
- 229920002101 Chitin Polymers 0.000 description 1
- 108090000288 Glycoproteins Proteins 0.000 description 1
- 102000003886 Glycoproteins Human genes 0.000 description 1
- 239000007995 HEPES buffer Substances 0.000 description 1
- 108090000604 Hydrolases Proteins 0.000 description 1
- 102000004157 Hydrolases Human genes 0.000 description 1
- 108090000723 Insulin-Like Growth Factor I Proteins 0.000 description 1
- 108090000542 Lymphotoxin-alpha Proteins 0.000 description 1
- 102000004083 Lymphotoxin-alpha Human genes 0.000 description 1
- LSDPWZHWYPCBBB-UHFFFAOYSA-N Methanethiol Chemical compound SC LSDPWZHWYPCBBB-UHFFFAOYSA-N 0.000 description 1
- 102000009664 Microtubule-Associated Proteins Human genes 0.000 description 1
- 108010020004 Microtubule-Associated Proteins Proteins 0.000 description 1
- 108010064696 N,O-diacetylmuramidase Proteins 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 108010038807 Oligopeptides Proteins 0.000 description 1
- 102000015636 Oligopeptides Human genes 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 101710176384 Peptide 1 Proteins 0.000 description 1
- 108010033276 Peptide Fragments Proteins 0.000 description 1
- 102000007079 Peptide Fragments Human genes 0.000 description 1
- 101800001386 Peptide II Proteins 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
- SOKRNBGSNZXYIO-UHFFFAOYSA-N Resinone Natural products CC(=C)C1CCC2(C)C(O)CC3(C)C(CCC4C5(C)CCC(=O)C(C)(C)C5CCC34C)C12 SOKRNBGSNZXYIO-UHFFFAOYSA-N 0.000 description 1
- 102000013275 Somatomedins Human genes 0.000 description 1
- 229920004890 Triton X-100 Polymers 0.000 description 1
- 239000013504 Triton X-100 Substances 0.000 description 1
- CPZHJYJSCCEDQX-UHFFFAOYSA-N [O]C1=CC=C([N+]([O-])=O)C=C1 Chemical compound [O]C1=CC=C([N+]([O-])=O)C=C1 CPZHJYJSCCEDQX-UHFFFAOYSA-N 0.000 description 1
- 230000003044 adaptive effect Effects 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000010100 anticoagulation Effects 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 230000006315 carbonylation Effects 0.000 description 1
- 238000005810 carbonylation reaction Methods 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 238000009833 condensation Methods 0.000 description 1
- 230000005494 condensation Effects 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 239000013632 covalent dimer Substances 0.000 description 1
- 238000013016 damping Methods 0.000 description 1
- 238000009792 diffusion process Methods 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 230000013595 glycosylation Effects 0.000 description 1
- 238000006206 glycosylation reaction Methods 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 230000000873 masking effect Effects 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 125000000325 methylidene group Chemical group [H]C([H])=* 0.000 description 1
- 238000003032 molecular docking Methods 0.000 description 1
- 150000002772 monosaccharides Chemical group 0.000 description 1
- VIKNJXKGJWUCNN-XGXHKTLJSA-N norethisterone Chemical compound O=C1CC[C@@H]2[C@H]3CC[C@](C)([C@](CC4)(O)C#C)[C@@H]4[C@@H]3CCC2=C1 VIKNJXKGJWUCNN-XGXHKTLJSA-N 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 230000001590 oxidative effect Effects 0.000 description 1
- 230000026731 phosphorylation Effects 0.000 description 1
- 238000006366 phosphorylation reaction Methods 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000000197 pyrolysis Methods 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 238000004007 reversed phase HPLC Methods 0.000 description 1
- 239000002795 scorpion venom Substances 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 238000004062 sedimentation Methods 0.000 description 1
- 238000002791 soaking Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 230000003595 spectral effect Effects 0.000 description 1
- 230000002269 spontaneous effect Effects 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- WROMPOXWARCANT-UHFFFAOYSA-N tfa trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F.OC(=O)C(F)(F)F WROMPOXWARCANT-UHFFFAOYSA-N 0.000 description 1
- HNKJADCVZUBCPG-UHFFFAOYSA-N thioanisole Chemical compound CSC1=CC=CC=C1 HNKJADCVZUBCPG-UHFFFAOYSA-N 0.000 description 1
- 238000001890 transfection Methods 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
- 238000002525 ultrasonication Methods 0.000 description 1
- 238000001291 vacuum drying Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Images
Landscapes
- Peptides Or Proteins (AREA)
Abstract
A protein preparation method provided by the invention comprises the following steps to connect a first peptide with hydrazide group on C terminus and a second peptide with cysteine on N terminus: mixing the first peptide and the second peptide in a solution containing NaNO2 and adjusting the mixed solution pH to an acidic pH so as to converting the hydrazide group of the first peptide into acyl azide group to obtain a first reaction mixture; adding a solution containing a reducing agent into the first reaction mixture and adjusting the pH to a neutral pH, wherein the reducing agent contains mercapto group, so as to convert the acyl azide group of the first peptide into thioester group, and maintaining the neutral pH so as to conducting a connection reaction between the thioester on the C terminus of the first peptide and the N terminus of the second peptide to produce proteins. A method for preparing polypeptide thioester compounds is also provided. According to the protein preparation method provided by the invention, proteins can be effectively synthesized.
Description
Technical field
The present invention relates to prepare protein and method of protein, relate more specifically to the method for attachment of peptide.
Background technology
The structure and function relation of protein is one of forward position hot issue of biology at present, and the acquisition of the protein that highly purified or specific non-natural is modified can promote the development in this field greatly.The chemosynthesis of protein and semi-synthetic be the important channel that is considered to obtain to have the target protein that ad hoc structure modifies, therefore develop efficient synthetic technology and have academic and commercial value.As far back as 1901, organic synthesis great master Fisher just proposed the conception of chemosynthetic organism enzyme.1963, Merrifield development solid-phase synthetic peptide technology came true the chemosynthetic organism bioactive peptide.Yet, to assemble very seriously on resin due to the long-chain polypeptide, solid-phase synthetic peptide is considered to realize the efficient synthetic of 50 following amino-acid residues.Based on this problem, the Kent of Chicago University develops nature chemistry connection strategy.Thereby this method can and realize that 100 above residues are amino acid whose synthetic with two sections above efficient connection of peptide fragment.At present, chemistry connects protein synthesis method that be considered to be most widely used and the most efficient naturally.The people such as Kent utilize its development " naturally chemistry connect " successfully to synthesize a series of protein molecules with natural function, generate albumen, HIV-1 proteolytic enzyme, human body N,O-Diacetylmuramidase, covalent dimer HIV-1 proteolytic enzyme, antifreeze protein, insulin-like growth factor etc. as Sigma I8405 inhibitor, the active II type of human secretory Phospholipid hydrolase, S type anticoagulation micro protein matter, cytopigment, the macromolecule modified artificial red cell of lymphotoxin albumen, homogeneous that contains 8 monosaccharide groups.
As shown in Figure 1, the elementary tactics of chemical connection method naturally: in the aqueous solution of neutrality, the ligation of efficient chemo-selective can occur in the peptide 2 of the thioesters compound of peptide 1 and N-end halfcystine.Yet naturally chemical interconnection technique need to prepare the thioesters compound of polypeptide.The thioesters compound of polypeptide need to adopt the Boc-SPPS method to synthesize, but Boc-SPPS need to use the HF of highly corrosive.In addition, glycosylation, phosphorylation and Sulfated polypeptide are unstable to HF, can not efficiently synthesize.Thereby present protein synthetic method haves much room for improvement.
Summary of the invention
The present invention is intended to solve at least one of technical problem that exists in prior art.For this reason, one object of the present invention is to propose a kind of protein synthetic method, can effectively carry out peptide and connect.
In one aspect of the invention, according to the embodiment of the present invention, a kind of method of protein for preparing is provided, and it comprises that the first peptide that through the following steps the C end is had the hydrazides group is connected for the second peptide of halfcystine with the N end: described the first peptide and described the second peptide are being contained NaNO
2Solution in mix and regulate pH to acid pH, obtain the first reaction mixture in order to the hydrazides group of described the first peptide is converted into the acid azide group; Add in described the first reaction mixture and contain the solution of reductive agent and regulate pH to neutral pH; wherein said reductive agent contains mercapto groups; so that the acid azide groups converted of described the first peptide is thioester group, keeps the C end thioesters of described neutral pH and then described the first peptide and the N end of described the second peptide ligation formation protein occurs.
According to feature of the present invention, the preparation method of protein can also have following additional technical feature:
According to one embodiment of present invention, described reductive agent is 4-sulfydryl phenylacetic acid.
According to one embodiment of present invention, to have the first peptide of hydrazides group be synthetic by the method that comprises the following steps to described C end: express the fusion rotein that the protein that contains intron and C hold the first peptide that there is no the hydrazides group; And described fusion rotein is carried out hydrazinolysis, obtain the first peptide that described C end has the hydrazides group.
According to one embodiment of present invention, described acid pH is 3.0-4.0.
According to one embodiment of present invention, utilize sodium hydroxide to regulate pH to described neutral pH.
In another aspect of the present invention, according to embodiments of the invention, also provide a kind of method for preparing the polypeptide thioesters compound, it is characterized in that comprising the following steps: will as the first peptide of raw material with contain NaNO
2Solution mix, and regulate pH to acid pH, obtain the first preparation feedback mixture in order to the hydrazides group of described the first peptide is converted into the acid azide group; Add the solution that contains reductive agent in described the first preparation feedback mixture, and regulate pH to neutral pH, wherein said reductive agent contains mercapto groups, so that the acid azide groups converted of described the first peptide is thioester group, forms the polypeptide thioesters compound.
According to embodiments of the invention, the method for preparing the polypeptide thioesters compound can also have other additional technical features:
According to one embodiment of present invention, described acid pH is 3.0-4.0.
According to one embodiment of present invention, described reductive agent is 4-sulfydryl phenylacetic acid.
According to one embodiment of present invention, to have the first peptide of hydrazides group be synthetic by the method that comprises the following steps to described C end: express the fusion rotein that the protein that contains intron and C hold the first peptide that there is no the hydrazides group; And described fusion rotein is carried out hydrazinolysis, obtain the first peptide that described C end has the hydrazides group.
According to one embodiment of present invention, utilize sodium hydroxide to regulate pH to described neutral pH.
The aspect that the present invention adds and advantage part in the following description provide, and part will become obviously from the following description, or recognize by practice of the present invention.
Description of drawings
Above-mentioned and/or the additional aspect of the present invention and advantage will become from the following description of the accompanying drawings of embodiments and obviously and easily understand, wherein:
Fig. 1 is for carrying out the reaction schematic diagram of protein synthesis according to the Nature Link method of prior art.
Fig. 2 is the reaction schematic diagram of protein synthetic method according to an embodiment of the invention.
Fig. 3-25 are respectively the product detected result according to embodiment of the present invention 3-23.Wherein,
Fig. 3 is the synthetic H-Leu-Tyr-Arg-Ala-Tyr-Cys-Lys-Tyr-Met-His-OH of polypeptide hydrazides method, and annotate: 1a is polypeptide hydrazides raw material, and 2 for the N end is the pentapeptide of halfcystine, and 3a is for connecting product;
Fig. 4 is the synthetic H-Leu-Tyr-Arg-Ala-Ala-Cys-Lys-Tyr-Met-His-OH of polypeptide hydrazides method, and annotate: 1b is polypeptide hydrazides raw material, and 2 are N end halfcystine pentapeptide, and 3b is for connecting product;
Fig. 5 is the synthetic H-Leu-Tyr-Arg-Ala-Met-Cys-Lys-Tyr-Met-His-OH of polypeptide hydrazides method, and annotate: 1c is polypeptide hydrazides raw material, and 2 for the N end is the pentapeptide of halfcystine, and 3c is for connecting product;
Fig. 6 is the synthetic H-Leu-Tyr-Arg-Ala-Phe-Cys-Lys-Tyr-Met-His-OH of polypeptide hydrazides method, and annotate: 1d is polypeptide hydrazides raw material, and 2 for the N end is the pentapeptide of halfcystine, and 3d is for connecting product;
Fig. 7 is the synthetic H-Leu-Tyr-Arg-Ala-Phe-Cys-Lys-Tyr-Gly-His-OH of polypeptide hydrazides method, and annotate: 1e is polypeptide hydrazides raw material, and 2 for the N end is the pentapeptide of halfcystine, and 3e is for connecting product;
Fig. 8 is the synthetic H-Leu-Tyr-Arg-Ala-His-Cys-Lys-Tyr-Gly-His-OH of polypeptide hydrazides method, and annotate: 1f is polypeptide hydrazides raw material, and 2 for the N end is the pentapeptide of halfcystine, and 3f is for connecting product;
Fig. 9 is the synthetic H-Leu-Tyr-Arg-Ala-Arg-Cys-Lys-Tyr-Gly-His-OH of polypeptide hydrazides method, and annotate: 1g is polypeptide hydrazides raw material, and 2 for the N end is the pentapeptide of halfcystine, and 3g is for connecting product;
Figure 10 is the synthetic H-Leu-Tyr-Arg-Ala-Ser-Cys-Lys-Tyr-Gly-His-OH of polypeptide hydrazides method, and annotate: 1h is polypeptide hydrazides raw material, and 2 for the N end is the pentapeptide of halfcystine, and 3h is for connecting product;
Figure 11 is the synthetic H-Leu-Tyr-Arg-Ala-Thr-Cys-Lys-Tyr-Gly-His-OH of polypeptide hydrazides method, and annotate: 1i is polypeptide hydrazides raw material, and 2 for the N end is the pentapeptide of halfcystine, and 3i is for connecting product;
Figure 12 is the synthetic H-Leu-Tyr-Arg-Ala-Cys-Cys-Lys-Tyr-Gly-His-OH of polypeptide hydrazides method, and annotate: 1j is polypeptide hydrazides raw material, and 2 for the N end is the pentapeptide of halfcystine, and 3j is for connecting product;
Figure 13 is the synthetic H-Leu-Tyr-Arg-Ala-Glu-Cys-Lys-Tyr-Gly-His-OH of polypeptide hydrazides method, and annotate: 1k is polypeptide hydrazides raw material, and 2 for the N end is the pentapeptide of halfcystine, and 3k is for connecting product;
Figure 14 is the synthetic H-Leu-Tyr-Arg-Ala-Lys-Cys-Lys-Tyr-Gly-His-OH of polypeptide hydrazides method, and annotate: 1l is polypeptide hydrazides raw material, and 2 for the N end is the pentapeptide of halfcystine, and 3l is for connecting product;
Figure 15 is the synthetic H-Leu-Tyr-Arg-Ala-Trp-Cys-Lys-Tyr-Gly-His-OH of polypeptide hydrazides method, and annotate: 1m is polypeptide hydrazides raw material, and 2 for the N end is the pentapeptide of halfcystine, and 3m is for connecting product;
Figure 16 is the synthetic H-Leu-Tyr-Arg-Ala-Leu-Cys-Lys-Tyr-Gly-His-OH of polypeptide hydrazides method, and annotate: 1n is polypeptide hydrazides raw material, and 2 for the N end is the pentapeptide of halfcystine, and 3n is for connecting product;
Figure 17 is the synthetic H-Leu-Tyr-Arg-Ala-Val-Cys-Lys-Tyr-Gly-His-OH of polypeptide hydrazides method, and annotate: 1o is polypeptide hydrazides raw material, and 2 for the N end is the pentapeptide of halfcystine, and 3o is for connecting product;
Figure 18 is the synthetic H-Leu-Tyr-Arg-Ala-Ile-Cys-Lys-Tyr-Gly-His-OH of polypeptide hydrazides method, and annotate: 1p is polypeptide hydrazides raw material, and 2 for the N end is the pentapeptide of halfcystine, and 3p is for connecting product;
Figure 19 is the synthetic H-Leu-Tyr-Arg-Ala-Pro-Cys-Lys-Tyr-Gly-His-OH of polypeptide hydrazides method, and annotate: 1q is polypeptide hydrazides raw material, and 2 for the N end is the pentapeptide of halfcystine, and 3q is for connecting product;
Figure 20 is the synthetic H-Leu-Tyr-Arg-Ser[β-D-Glc (OAc) 4 of polypeptide hydrazides method]-Ala--Cys-Lys-Tyr-Gly-His-OH,
Annotate: 2 is the pentapeptide of halfcystine for N holds
Figure 21 is the synthetic Trifolitoxin[Ala23Cys of polypeptide hydrazides method];
Figure 22 is the ligation of the polypeptide hydrazides method Css I I-the first step;
Figure 23 is the ligation of polypeptide hydrazides method Css I I-second step;
Figure 24 is polypeptide hydrazides method Css I I-the 3rd step ligation;
Figure 25 is semi-synthetic LC3, and annotate: a is the SDS-PAGE electrophoresis of the LC3 hydrazides of EPL protein expression; B, c are HPLC before and after protein connects; D, e are respectively the LC3 hydrazides and the ESI-MS that is connected product of expression.
Embodiment
Below with reference to accompanying drawing, embodiments of the invention are discussed.Yet those skilled in the art are to be understood that the embodiment about these accompanying drawings given here is for illustrative purposes, because the present invention extends on these limited embodiment.For example, those skilled in the art will be appreciated that the needs according to application-specific under teaching of the present invention, except the particular implementation of describing in embodiment below and illustrating is selected, can also use various substituting and proper method.That is, have multiple modification of the present invention and variation, the number of these modification and variation is too many and can not list, but within all fall into scope of the present invention.
Should further understand and the invention is not restricted to ad hoc approach described herein, compound, material, manufacturing technology, use and application, because these can change.Be to be understood that also term used herein is only for the purpose of describing specific embodiment, and be not intended to limit scope of the present invention.Must be noted that " " of singulative, " one ", " being somebody's turn to do " comprise plural reference, unless context explicitly points out reverse situation as using here and in claims.Thereby for example, quoting of " element " is quoting and comprising and being equal to well known to a person skilled in the art one or more elements.Similarly, another example, quoting of " step " or " device " is quoting and can comprise substep and sub-device one or more steps or device.All conjunctions that use should the highlyest comprise that mode understands with possible.Thereby the word "or" is appreciated that the definition with logical "or", rather than has the definition of logic " repel or ", unless context explicitly points out reverse situation.Structure described herein is appreciated that the functional equivalent that also refers to this structure.Can be understood to represent that the language that is similar to should so be understood, unless context explicitly points out reverse situation.
Unless otherwise defined, all technology used herein and scientific terminology have the common equivalent of understanding of those skilled in the art.Preferred method, technology, equipment and material have been described, although can use and those similar or be equal to any method, technology, equipment or materials described herein in practice of the present invention or test.Structure described herein also is appreciated that the functional equivalent that refers to this structure.Describe the present invention in detail referring now to the embodiment as explanation in the accompanying drawings.
By reading this announcement, those skilled in the art will apparent other variation and modification.Such variation and modification can relate to known in the art being equal to and further feature, and can substitute use or be increased to the feature of having described here.
Although in this application, claim is constructed to the particular combination of feature, the scope that is to be understood that announcement of the present invention also comprises clear and definite or implicit any new feature or any new combination of feature or any summary of feature of disclosing here, and no matter whether it relates to the identical invention of advocating with current any claim and whether alleviate as the present invention any or all identical technical problem regardless of it.
During the feature of describing in the context that separates embodiment also may be provided in the combination of single embodiment.On the contrary, the various features of describing in the context of single embodiment for simple and clear purpose also can provide separatedly or be provided in any suitable sub-portfolio.Applicant thereby remind in the application or the checking process according to any continuation application of the application may be to the new claim of composite construction of such feature and/or such feature.
As known for those skilled in the art, when the optimum manufacturing (particularly embodiments of the invention) that the business that designs any system is implemented, usually must carry out consideration and the compromise of many carefulnesses.Can configure according to the needs of application-specific according to the business of spirit of the present invention and teaching and implement, thereby to those skilled in the art by usual technical ability and the known technology that uses them can suitably omit, comprise, any aspect, feature, function, result, parts, method or the step of adaptive, mixing and coupling or improvement and/or the optimization teaching relevant to the embodiment of any description of the present invention, implement with the expectation of the needs of realizing solving application-specific.
Detailed description of preferred embodiment is provided here.Yet, be to be understood that the present invention can embodied in various forms realize.Thereby, here the specific detail that discloses is not appreciated that restriction, but should be understood to as the basis of claim and as being used for teaching those skilled in the art any suitable detailed system, structure or mode employing expression of the present invention basis actually.
In this article, term " first " and " second " are only for polypeptide is distinguished, and do not have any meaning about precedence relationship.
The term that uses in this article " polypeptide " can be replaced use mutually with " protein ", its implication should be made broad understanding, the compound that refers to any amino acid monomer to link together with peptide chain and form, also may contain some amino acid analogue, wherein amino acid monomer in polypeptide or protein number and be not particularly limited, sometimes also can become " oligopeptides ".
In this article, term " N holds WPH " and " N end be the peptide of halfcystine " can Alternates, all refer to the peptide that N-terminal is halfcystine, and the sulfydryl of halfcystine are not protected.
In one aspect of the invention, according to the embodiment of the present invention, provide a kind of method of protein for preparing, it comprises that the first peptide is connected with the second peptide, and wherein the C of the first peptide end has the hydrazides group, and the N end of the second peptide is halfcystine.Particularly, the first peptide is comprised with the process that the second peptide is connected:
At first, the hydrazides group that comprises in the C end with the first peptide is converted into the acid azide group, and is concrete, and the first peptide and the second peptide are being contained NaNO
2Solution in mix and regulate pH to acid pH (in one embodiment of the invention, this acid pH is 3.0-4.0), obtain the first reaction mixture in order to the hydrazides group of described the first peptide is converted into the acid azide group.According to embodiments of the invention, C end has the first peptide of hydrazides group can be by utilizing the Wang resin as the carrier solid phase synthesis.This has been avoided adopting in the synthetic thioesters compound of available technology adopting Boc-SPPS method the drawback of HF.In addition, according to embodiments of the invention, can also adopt the synthetic C end of the method that comprises the following step to have the first peptide of hydrazides group: expression contains the protein of intron (Intein) and the fusion rotein that the C end does not have the first peptide of hydrazides group; And described fusion rotein is carried out hydrazinolysis, obtain the first peptide that described C end has the hydrazides group.Thus, can avoid adopting the drawback of HF in the synthetic thioesters compound of available technology adopting Boc-SPPS method, and can utilize biosynthetic method synthetic peptide according to embodiments of the invention, thus, can enhance productivity.According to embodiments of the invention, N-terminal is that the second peptide of halfcystine can synthesize by any known method, for example synthesize by the method for biological expression, concrete, can be by for example changing the DNA fragmentation of the specific peptide sequence of coding to microorganism in intestinal bacteria over to or containing the carrier of this DNA fragmentation, and be suitable for cultivating under the condition that described DNA fragmentation expresses described through the microorganism that transforms intestinal bacteria for example, next expressed polypeptide is separated and purifying.
next, with the acid azide groups converted be the mercapto ester group and and then by the spontaneous reaction between the sulfydryl of mercapto ester group and halfcystine, the first peptide is being connected with the second peptide under pH neutral, concrete, add in the first reaction mixture that obtains previously and contain reductive agent (according to one embodiment of present invention, this reductive agent be the solution of MPAA (4-sulfydryl phenylacetic acid) and regulate pH to neutral pH (according to one embodiment of present invention, adopt sodium hydroxide to carry out pH regulator), wherein said reductive agent contains mercapto groups, so that the acid azide groups converted of described the first peptide is thioester group, keep the C end thioesters of described neutral pH and then described the first peptide and the N end of described the second peptide ligation formation protein occurs.
This shows, prepare according to an embodiment of the invention method of protein, being the polypeptide chain connection technology take the synthetic polypeptide thioesters of Boc forensic chemistry as primitive unit not, is the novel polypeptide interconnection technique of raw material and adopt the hydrazides compound of chemosynthesis milli polypeptide with no difficulty.The connection procedure that relates in the present invention comprises two steps: the first step, the hydrazides compound of polypeptide by NaNO
2Be oxidized to polypeptide acid azide (oxidizing condition: pH 3.0～4.0, NaNO
2(10mM), 20.0min); Second step, under the mercaptan of a large amount of reductibilities exists, excessive oxygenant (NaNO
2) be consumed, the polypeptide acyl group is converted into polypeptide thioesters, and the polypeptide thioesters of formation and the reaction of N-end WPH selectivity efficient realize the docking of polypeptide fragment.Key of the present invention is by oxidising process, high stability polypeptide hydrazides to be converted into the high reactivity polypeptide acid azide that can be used for connection.The polypeptide hydrazides is very stable, and is all stable to piperidines, TFA and HF, therefore can simply and efficiently prepare by Fmoc or Boc method, can be used for the synthetic of glycoprotein.In addition, aforementioned polypeptides hydrazides-redox ligation can repeat, to realize the connection of many polypeptide.
In addition, according to embodiments of the invention, also provide a kind of method for preparing the polypeptide thioesters compound, it is characterized in that comprising the following steps:
At first, the hydrazides group that comprises in the C of the first peptide end is converted into the acid azide group, concrete, will as the first peptide of raw material with contain NaNO
2Solution mix, and regulate pH to acid pH (in one embodiment of the invention, this acid pH is 3.0-4.0), obtain the first preparation feedback mixture in order to the hydrazides group of described the first peptide is converted into the acid azide group.According to embodiments of the invention, C end has the first peptide of hydrazides group can be by utilizing the Wang resin as the carrier solid phase synthesis.This has been avoided adopting in the synthetic thioesters compound of available technology adopting Boc-SPPS method the drawback of HF.In addition, according to embodiments of the invention, can also adopt the synthetic C end of the method that comprises the following step to have the first peptide of hydrazides group: expression contains the protein of intron (Intein) and the fusion rotein that the C end does not have the first peptide of hydrazides group; And described fusion rotein is carried out hydrazinolysis, obtain the first peptide that described C end has the hydrazides group.Thus, can avoid adopting the drawback of HF in the synthetic thioesters compound of available technology adopting Boc-SPPS method, and can utilize biosynthetic method synthetic peptide according to embodiments of the invention, thus, can enhance productivity.
Then; add in described the first preparation feedback mixture and contain reductive agent (according to one embodiment of present invention; this reductive agent is the solution of MPAA (4-sulfydryl phenylacetic acid); and regulate pH to neutral pH (according to one embodiment of present invention; adopt sodium hydroxide to carry out pH regulator); wherein said reductive agent contains mercapto groups, so that the acid azide groups converted of described the first peptide is thioester group, forms the polypeptide thioesters compound.
The below provides some embodiment so that further the present invention will be described, and these embodiment also limit the scope of the invention by any way.
For clearer, subsequent embodiment is described, the below provides the symbol abbreviation expression of each raw material of using in each embodiment, and in each embodiment, the place of using the symbol abbreviation all refers to content corresponding to corresponding symbol abbreviation, and is specific as follows:
The DCM methylene dichloride
DCM/DMF/MeOH/DCM methylene dichloride/DMF/ethanol/methylene
HBTU benzotriazole-N, N, N ', N '-tetramethyl-urea phosphofluoric acid ester
The HOBt I-hydroxybenzotriazole
The DIEA diisopropylethylamine
DMAP N, N-dimethyl-4-pyridine amine
The TFA trifluoroacetic acid
The EDT 1，2-ethandithiol
1. with Wang resin (1.5 gram) DCM (14mL) swelling, pack in round-bottomed flask, under cryosel is bathed and is stirred, add successively N-methylmorpholine (0.16mL) and p-nitrophenyl oxygen acyl chlorides (0.29 gram), after the system stirring at normal temperature is spent the night, resin transfer is washed resin with a large amount of DCM/DMF/MeOH/DCM to sintered filter funnel, with resin and vacuum drier dried overnight.
2. the Wang resin (buying in Tianjin Nankai Hecheng S﹠T Co., Ltd.) of p-nitrophenyl oxygen carbonylation is packed in round-bottomed flask, (form: hydrazine hydrate: DMF: DCM=0.16mL: 30mL: 22mL), stirring at normal temperature is spent the night to add the pre-cooled mixed solution that configures.Resin transfer is washed resin with a large amount of DCM/DMF/MeOH/DCM to sintered filter funnel, with resin and vacuum drier dried overnight.Namely get the hydrazides resin for synthetic polypeptide hydrazides.
1. the hydrazides resin is added solid phase reactor with the DMF/DCM swelling of 1: 13 hours; now configure mixed solution (the target peptide C of the HBTU:4 equivalent HOBt:8 equivalent DIEA:0.1 equivalent DMAP:4 equivalent protection of 3.6 equivalents holds first amino acid) and added resin reaction 8 hours, resin is washed with a large amount of DMF, DCM, DMF successively.Masking reagent (acetic anhydride: DIEA: DMF=1: 1: 8) soaked resin after 10 minutes, again once used a large amount of DMF, DCM, DMF washing resin.Add the DMF solution soaking resin 5 minutes and 10 minutes of 20% piperidines.Added resin reaction 60 minutes with the mixed solution that adds existing preparation after a large amount of DMF, DCM, DMF washing (second amino acid of target peptide C end of the HBTU:4 equivalent HOBt:8 equivalent DIEA:4 equivalent protection of 3.6 equivalents).After a large amount of DMF, DCM, DMF washing resin, with the DMF solution-treated of 20% piperidines 5 minutes and 10 minutes.Operation above amino acid whose condensation under connecing repeats.
2. after the target peptide solid phase synthesis finished, respectively with a large amount of DMF, DCM washing, after vacuum-drying, (85%TFA: 5% thioanisole: 5%EDT: 2.5% phenol: 2.5% water) 5-10mL processed 2-3 hour to add acid cutting reagent with resin.Concentrated acidic contains the acid cutting reagent of target polypeptides, adds the ether sedimentation of 20 equivalents, centrifugal Powdered just peptide.Preparation HPLC purification of target peptide (mass spectrum is determined), the highly purified target polypeptides hydrazides of vacuum lyophilization.The polypeptide hydrazides that is used for connecting that relates to later all obtains with this method, and common polypeptide is to adopt Wang resin or the preparation of Rink amide resin Fmoc solid phase synthesis.
Embodiment 3, the synthetic H-Leu-Tyr-Arg-Ala-Tyr-Cys-Lys-Tyr-Met-His-OH of polypeptide hydrazides method
1. weigh polypeptide hydrazides H-Leu-Tyr-Arg-Ala-Tyr-NHNH
2(1.5 milligrams), H-Cys-Lys-Tyr-Met-His-OH (1.95 milligrams) are dissolved in 0.65mLPBS (pH 3.0 for 6.0M Guanidinium hydrochloride, 0.2M phosphoric acid salt), add 50uL benzamide solution (40mM).
2. get the 0.25mL mentioned solution and put into the vial of 1.5mL, add magneton, (approximately-10 ℃ of left and right) stirs the lower 25uLNaNO of dropping in cryosel is bathed
2(0.2M) aqueous solution, low-temp reaction is about 20 minutes.
3. after oxidation finishes, drip 0.25mLMPAA guanidine hydrochloride solution (0.2MMPAA, 6.0M Guanidinium hydrochloride, 0.2M phosphoric acid salt, pH 5.0), remove cryosel and bathe, get the acidity of the careful conditioned reaction liquid of NaOH (2.0M) solution to neutral (acidity is measured with the micro glass pH meter).
4. after reacting 2 hours, get the 20uL reaction solution and mixed 5 minutes with 60uL TCEP (30mM), HPLC detects, and separates the peak that makes new advances, and mass spectrum is defined as connecting product, and result as shown in Figure 3.
Embodiment 4, the synthetic H-Leu-Tyr-Arg-Ala-Ala-Cys-Lys-Tyr-Met-His-OH of polypeptide hydrazides method
1. weigh polypeptide hydrazides H-Leu-Tyr-Arg-Ala-Ala-NHNH
2(1.3 milligrams), H-Cys-Lys-Tyr-Met-His-OH (1.95 milligrams) are dissolved in 0.65mLPBS (pH 3.0 for 6.0M Guanidinium hydrochloride, 0.2M phosphoric acid salt), add 50uL benzamide solution (40mM).
2. get the 0.25mL mentioned solution and put into the vial of 1.5mL, add magneton, (approximately-10 ℃ of left and right) stirs the lower 25uLNaNO of dropping in cryosel is bathed
2(0.2M) aqueous solution, low-temp reaction is about 20 minutes.
3. after oxidation finishes, drip 0.25mLMPAA guanidine hydrochloride solution (0.2MMPAA, 6.0M Guanidinium hydrochloride, 0.2M phosphoric acid salt, pH 5.0), remove cryosel and bathe, get the acidity of the careful conditioned reaction liquid of NaOH (2.0M) solution to neutral (acidity is measured with the micro glass pH meter).
4. after reacting 2 hours, get the 20uL reaction solution and mixed 5 minutes with 60uL TCEP (30mM), HPLC detects, and separates the peak that makes new advances, and mass spectrum is defined as connecting product, and result as shown in Figure 4.
1. weigh polypeptide hydrazides H-Leu-Tyr-Arg-Ala-Met-NHNH
2(1.4 milligrams), H-Cys-Lys-Tyr-Met-His-OH (1.95 milligrams) are dissolved in 0.65mLPBS (pH 3.0 for 6.0M Guanidinium hydrochloride, 0.2M phosphoric acid salt), add 50uL benzamide solution (40mM).
2. get the 0.25mL mentioned solution and put into the vial of 1.5mL, add magneton, (about approximately-10oC) stirs the lower 25uLNaNO of dropping in cryosel is bathed
2(0.2M) aqueous solution, low-temp reaction is about 20 minutes.
3. after oxidation finishes, drip 0.25mLMPAA guanidine hydrochloride solution (0.2MMPAA, 6.0M Guanidinium hydrochloride, 0.2M phosphoric acid salt, pH 5.0), remove cryosel and bathe, get the acidity of the careful conditioned reaction liquid of NaOH (2.0M) solution to neutral (acidity is measured with the micro glass pH meter).
4. after reacting 2 hours, get the 20uL reaction solution and mixed 5 minutes with 60uL TCEP (30mM), HPLC detects, and separates the peak that makes new advances, and mass spectrum is defined as connecting product, and result as shown in Figure 5.
1. weigh polypeptide hydrazides H-Leu-Tyr-Arg-Ala-Phe-NHNH
2(1.5 milligrams), H-Cys-Lys-Tyr-Met-His-OH (1.95 milligrams) are dissolved in 0.65mLPBS (pH 3.0 for 6.0M Guanidinium hydrochloride, 0.2M phosphoric acid salt), add 50uL benzamide solution (40mM).
2. get the 0.25mL mentioned solution and put into the vial of 1.5mL, add magneton, (approximately-10 ℃ of left and right) stirs the lower 25uLNaNO of dropping in cryosel is bathed
2(0.2M) aqueous solution, low-temp reaction is about 20 minutes.
3. after oxidation finishes, drip 0.25mLMPAA guanidine hydrochloride solution (0.2MMPAA, 6.0M Guanidinium hydrochloride, 0.2M phosphoric acid salt, pH 5.0), remove cryosel and bathe, get the acidity of the careful conditioned reaction liquid of NaOH (2.0M) solution to neutral (acidity is measured with the micro glass pH meter).
4. after reacting 2 hours, get the 20uL reaction solution and mixed 5 minutes with 60uL TCEP (30mM), HPLC detects, and separates the peak that makes new advances, and mass spectrum is defined as connecting product, and result as shown in Figure 6.
1. weigh polypeptide hydrazides H-Leu-Tyr-Arg-Ala-Gly-NHNH
2(1.3 milligrams), H-Cys-Lys-Tyr-Met-His-OH (1.95 milligrams) are dissolved in 0.65mLPBS (pH 3.0 for 6.0M Guanidinium hydrochloride, 0.2M phosphoric acid salt), add 50uL benzamide solution (40mM).
2. get the 0.25mL mentioned solution and put into the vial of 1.5mL, add magneton, (approximately-10 ℃ of left and right) stirs the lower 25uLNaNO of dropping in cryosel is bathed
2(0.2M) aqueous solution, low-temp reaction is about 20 minutes.
3. after oxidation finishes, drip 0.25mLMPAA guanidine hydrochloride solution (0.2MMPAA, 6.0M Guanidinium hydrochloride, 0.2M phosphoric acid salt, pH 5.0), remove cryosel and bathe, get the acidity of the careful conditioned reaction liquid of NaOH (2.0M) solution to neutral (acidity is measured with the micro glass pH meter).
4. after reacting 2 hours, get the 20uL reaction solution and mixed 5 minutes with 60uL TCEP (30mM), HPLC detects, and separates the peak that makes new advances, and mass spectrum is defined as connecting product, and result as shown in Figure 7.
2. get the 0.25mL mentioned solution and put into the vial of 1.5mL, add magneton, (approximately-10 ℃ of left and right) stirs the lower 25uLNaNO of dropping in cryosel is bathed
2(0.2M) aqueous solution, low-temp reaction is about 20 minutes.
3. after oxidation finishes, drip 0.25mLMPAA guanidine hydrochloride solution (0.2MMPAA, 6.0M Guanidinium hydrochloride, 0.2M phosphoric acid salt, pH 5.0), remove cryosel and bathe, get the acidity of the careful conditioned reaction liquid of NaOH (2.0M) solution to neutral (acidity is measured with the micro glass pH meter).
4. after reacting 2 hours, get the 20uL reaction solution and mixed 5 minutes with 60uL TCEP (30mM), HPLC detects, and separates the peak that makes new advances, and mass spectrum is defined as connecting product, and result as shown in Figure 8.
1. weigh polypeptide hydrazides H-Leu-Tyr-Arg-Ala-Arg-NHNH
2(1.5 milligrams), H-Cys-Lys-Tyr-Met-His-OH (1.95 milligrams) are dissolved in 0.65mLPBS (pH 3.0 for 6.0M Guanidinium hydrochloride, 0.2M phosphoric acid salt), add 50uL benzamide solution (40mM).
2. get the 0.25mL mentioned solution and put into the vial of 1.5mL, add magneton, (approximately-10 ℃ of left and right) stirs the lower 25uLNaNO of dropping in cryosel is bathed
2(0.2M) aqueous solution, low-temp reaction is about 20 minutes.
3. after oxidation finishes, drip 0.25mLMPAA guanidine hydrochloride solution (0.2MMPAA, 6.0M Guanidinium hydrochloride, 0.2M phosphoric acid salt, pH 5.0), remove cryosel and bathe, get the acidity of the careful conditioned reaction liquid of NaOH (2.0M) solution to neutral (acidity is measured with the micro glass pH meter).
4. after reacting 2 hours, get the 20uL reaction solution and mixed 5 minutes with 60uL TCEP (30mM), HPLC detects, and separates the peak that makes new advances, and mass spectrum is defined as connecting product, and result as shown in Figure 9.
1. weigh polypeptide hydrazides H-Leu-Tyr-Arg-Ala-Ser-NHNH
2(1.3 milligrams), H-Cys-Lys-Tyr-Met-His-OH (1.95 milligrams) are dissolved in 0.65mLPBS (pH 3.0 for 6.0M Guanidinium hydrochloride, 0.2M phosphoric acid salt), add 50uL benzamide solution (40mM).
2. get the 0.25mL mentioned solution and put into the vial of 1.5mL, add magneton, (approximately-10 ℃ of left and right) stirs the lower 25uLNaNO of dropping in cryosel is bathed
2(0.2M) aqueous solution, low-temp reaction is about 20 minutes.
3. after oxidation finishes, drip 0.25mLMPAA guanidine hydrochloride solution (0.2MMPAA, 6.0M Guanidinium hydrochloride, 0.2M phosphoric acid salt, pH 5.0), remove cryosel and bathe, get the acidity of the careful conditioned reaction liquid of NaOH (2.0M) solution to neutral (acidity is measured with the micro glass pH meter).
4. after reacting 2 hours, get the 20uL reaction solution and mixed 5 minutes with 60uL TCEP (30mM), HPLC detects, and separates the peak that makes new advances, and mass spectrum is defined as connecting product, and result as shown in figure 10.
1. weigh polypeptide hydrazides H-Leu-Tyr-Arg-Ala-Thr-NHNH
2(1.4 milligrams), H-Cys-Lys-Tyr-Met-His-OH (1.95 milligrams) are dissolved in 0.65mLPBS (pH 3.0 for 6.0M Guanidinium hydrochloride, 0.2M phosphoric acid salt), add 50uL benzamide solution (40mM).
2. get the 0.25mL mentioned solution and put into the vial of 1.5mL, add magneton, (approximately-10 ℃ of left and right) stirs the lower 25uLNaNO of dropping in cryosel is bathed
2(0.2M) aqueous solution, low-temp reaction is about 20 minutes.
3. after oxidation finishes, drip 0.25mLMPAA guanidine hydrochloride solution (0.2MMPAA, 6.0M Guanidinium hydrochloride, 0.2M phosphoric acid salt, pH 5.0), remove cryosel and bathe, get the acidity of the careful conditioned reaction liquid of NaOH (2.0M) solution to neutral (acidity is measured with the micro glass pH meter).
4. after reacting 2 hours, get the 20uL reaction solution and mixed 5 minutes with 60uL TCEP (30mM), HPLC detects, and separates the peak that makes new advances, and mass spectrum is defined as connecting product, and result as shown in figure 11.
Embodiment 12, the synthetic H-Leu-Tyr-Arg-Ala-Cys-Cys-Lys-Tyr-Met-His-OH of polypeptide hydrazides method
1. weigh polypeptide hydrazides H-Leu-Tyr-Arg-Ala-Cys-NHNH
2(1.4 milligrams), H-Cys-Lys-Tyr-Met-His-OH (1.95 milligrams) are dissolved in 0.65mLPBS (pH 3.0 for 6.0M Guanidinium hydrochloride, 0.2M phosphoric acid salt), add 50uL benzamide solution (40mM).
2. get the 0.25mL mentioned solution and put into the vial of 1.5mL, add magneton, (approximately-10 ℃ of left and right) stirs the lower 25uLNaNO of dropping in cryosel is bathed
2(0.2M) aqueous solution, low-temp reaction is about 20 minutes.
3. after oxidation finishes, drip 0.25mLMPAA guanidine hydrochloride solution (0.2MMPAA, 6.0M Guanidinium hydrochloride, 0.2M phosphoric acid salt, pH 5.0), remove cryosel and bathe, get the acidity of the careful conditioned reaction liquid of NaOH (2.0M) solution to neutral (acidity is measured with the micro glass pH meter).
4. after reacting 2 hours, get the 20uL reaction solution and mixed 5 minutes with 60uL TCEP (30mM), HPLC detects, and separates the peak that makes new advances, and mass spectrum is defined as connecting product, and result as shown in figure 12.
Embodiment 13, the synthetic H-Leu-Tyr-Arg-Ala-Glu-Cys-Lys-Tyr-Met-His-OH of polypeptide hydrazides method
1. weigh polypeptide hydrazides H-Leu-Tyr-Arg-Ala-Glu-NHNH
2(1.4 milligrams), H-Cys-Lys-Tyr-Met-His-OH (1.95 milligrams) are dissolved in 0.65mLPBS (pH 3.0 for 6.0M Guanidinium hydrochloride, 0.2M phosphoric acid salt), add 50uL benzamide solution (40mM).
2. get the 0.25mL mentioned solution and put into the vial of 1.5mL, add magneton, (approximately-10 ℃ of left and right) stirs the lower 25uLNaNO of dropping in cryosel is bathed
2(0.2M) aqueous solution, low-temp reaction is about 20 minutes.
3. after oxidation finishes, drip 0.25mLMPAA guanidine hydrochloride solution (0.2MMPAA, 6.0M Guanidinium hydrochloride, 0.2M phosphoric acid salt, pH 5.0), remove cryosel and bathe, get the acidity of the careful conditioned reaction liquid of NaOH (2.0M) solution to neutral (acidity is measured with the micro glass pH meter).
4. after reacting 2 hours, get the 20uL reaction solution and mixed 5 minutes with 60uL TCEP (30mM), HPLC detects, and separates the peak that makes new advances, and mass spectrum is defined as connecting product, and result as shown in figure 13.
1. weigh polypeptide hydrazides H-Leu-Tyr-Arg-Ala-Lys-NHNH
2(1.4 milligrams), H-Cys-Lys-Tyr-Met-His-OH (1.95 milligrams) are dissolved in 0.65mLPBS (pH 3.0 for 6.0M Guanidinium hydrochloride, 0.2M phosphoric acid salt), add 50uL benzamide solution (40mM).
2. get the 0.25mL mentioned solution and put into the vial of 1.5mL, add magneton, (approximately-10 ℃ of left and right) stirs the lower 25uLNaNO of dropping in cryosel is bathed
2(0.2M) aqueous solution, low-temp reaction is about 20 minutes.
3. after oxidation finishes, drip 0.25mLMPAA guanidine hydrochloride solution (0.2MMPAA, 6.0M Guanidinium hydrochloride, 0.2M phosphoric acid salt, pH 5.0), remove cryosel and bathe, get the acidity of the careful conditioned reaction liquid of NaOH (2.0M) solution to neutral (acidity is measured with the micro glass pH meter).
4. after reacting 2 hours, get the 20uL reaction solution and mixed 5 minutes with 60uL TCEP (30mM), HPLC detects, and separates the peak that makes new advances, and mass spectrum is defined as connecting product, and result as shown in figure 14.
Embodiment 15, the synthetic H-Leu-Tyr-Arg-Ala-Trp-Cys-Lys-Tyr-Met-His-OH of polypeptide hydrazides method
1. weigh polypeptide hydrazides H-Leu-Tyr-Arg-Ala-Trp-NHNH
2(1.5 milligrams), H-Cys-Lys-Tyr-Met-His-OH (1.95 milligrams) are dissolved in 0.65mLPBS (pH 3.0 for 6.0M Guanidinium hydrochloride, 0.2M phosphoric acid salt), add 50uL benzamide solution (40mM).
2. get the 0.25mL mentioned solution and put into the vial of 1.5mL, add magneton, (approximately-10 ℃ of left and right) stirs the lower 25uLNaNO of dropping in cryosel is bathed
2(0.2M) aqueous solution, low-temp reaction is about 20 minutes.
3. after oxidation finishes, drip 0.25mLMPAA guanidine hydrochloride solution (0.2MMPAA, 6.0M Guanidinium hydrochloride, 0.2M phosphoric acid salt, pH 5.0), remove cryosel and bathe, get the acidity of the careful conditioned reaction liquid of NaOH (2.0M) solution to neutral (acidity is measured with the micro glass pH meter).
4. after reacting 2 hours, get the 20uL reaction solution and mixed 5 minutes with 60uL TCEP (30mM), HPLC detects, and separates the peak that makes new advances, and mass spectrum is defined as connecting product, and result as shown in figure 15.
Embodiment 16, the synthetic H-Leu-Tyr-Arg-Ala-Leu-Cys-Lys-Tyr-Met-His-OH of polypeptide hydrazides method
1. weigh polypeptide hydrazides H-Leu-Tyr-Arg-Ala-Leu-NHNH
2(1.4 milligrams), H-Cys-Lys-Tyr-Met-His-OH (1.95 milligrams) are dissolved in 0.65mLPBS (pH 3.0 for 6.0M Guanidinium hydrochloride, 0.2M phosphoric acid salt), add 50uL benzamide solution (40mM).
2. get the 0.25mL mentioned solution and put into the vial of 1.5mL, add magneton, (approximately-10 ℃ of left and right) stirs the lower 25uLNaNO of dropping in cryosel is bathed
2(0.2M) aqueous solution, low-temp reaction is about 20 minutes.
3. after oxidation finishes, drip 0.25mLMPAA guanidine hydrochloride solution (0.2MMPAA, 6.0M Guanidinium hydrochloride, 0.2M phosphoric acid salt, pH 5.0), remove cryosel and bathe, get the acidity of the careful conditioned reaction liquid of NaOH (2.0M) solution to neutral (acidity is measured with the micro glass pH meter).
4. after reacting 2 hours, get the 20uL reaction solution and mixed 5 minutes with 60uL TCEP (30mM), HPLC detects, and separates the peak that makes new advances, and mass spectrum is defined as connecting product, and result as shown in figure 16.
Embodiment 17, the synthetic H-Leu-Tyr-Arg-Ala-Val-Cys-Lys-Tyr-Met-His-OH of polypeptide hydrazides method
1. weigh polypeptide hydrazides H-Leu-Tyr-Arg-Ala-Val-NHNH
2(1.3 milligrams), H-Cys-Lys-Tyr-Met-His-OH (1.95 milligrams) are dissolved in 0.65mLPBS (pH 3.0 for 6.0M Guanidinium hydrochloride, 0.2M phosphoric acid salt), add 50uL benzamide solution (40mM).
2. get the 0.25mL mentioned solution and put into the vial of 1.5mL, add magneton, (approximately-10 ℃ of left and right) stirs the lower 25uLNaNO of dropping in cryosel is bathed
2(0.2M) aqueous solution, low-temp reaction is about 20 minutes.
3. after oxidation finishes, drip 0.25mLMPAA guanidine hydrochloride solution (0.2MMPAA, 6.0M Guanidinium hydrochloride, 0.2M phosphoric acid salt, pH 5.0), remove cryosel and bathe, get the acidity of the careful conditioned reaction liquid of NaOH (2.0M) solution to neutral (acidity is measured with the micro glass pH meter).
4. after reacting 2 hours, get the 20uL reaction solution and mixed 5 minutes with 60uL TCEP (30mM), HPLC detects, and separates the peak that makes new advances, and mass spectrum is defined as connecting product, and result as shown in figure 17.
Embodiment 18, the synthetic H-Leu-Tyr-Arg-Ala-Ile-Cys-Lys-Tyr-Met-His-OH of polypeptide hydrazides method
1. weigh polypeptide hydrazides H-Leu-Tyr-Arg-Ala-Ile-NHNH
2(1.4 milligrams), H-Cys-Lys-Tyr-Met-His-OH (1.95 milligrams) are dissolved in 0.65mLPBS (pH 3.0 for 6.0M Guanidinium hydrochloride, 0.2M phosphoric acid salt), add 50uL benzamide solution (40mM).
2. get the 0.25mL mentioned solution and put into the vial of 1.5mL, add magneton, (approximately-10 ℃ of left and right) stirs the lower 25uLNaNO of dropping in cryosel is bathed
2(0.2M) aqueous solution, low-temp reaction is about 20 minutes.
3. after oxidation finishes, drip 0.25mLMPAA guanidine hydrochloride solution (0.2MMPAA, 6.0M Guanidinium hydrochloride, 0.2M phosphoric acid salt, pH 5.0), remove cryosel and bathe, get the acidity of the careful conditioned reaction liquid of NaOH (2.0M) solution to neutral (acidity is measured with the micro glass pH meter).
4. after reacting 2 hours, get the 20uL reaction solution and mixed 5 minutes with 60uL TCEP (30mM), HPLC detects, and separates the peak that makes new advances, and mass spectrum is defined as connecting product, and result as shown in figure 18.
1. weigh polypeptide hydrazides H-Leu-Tyr-Arg-Ala-Pro-NHNH
2(1.3 milligrams), H-Cys-Lys-Tyr-Met-His-OH (1.95 milligrams) are dissolved in 0.65mLPBS (pH 3.0 for 6.0M Guanidinium hydrochloride, 0.2M phosphoric acid salt), add 50uL benzamide solution (40mM).
2. get the 0.25mL mentioned solution and put into the vial of 1.5mL, add magneton, (approximately-10 ℃ of left and right) stirs the lower 25uLNaNO of dropping in cryosel is bathed
2(0.2M) aqueous solution, low-temp reaction is about 20 minutes.
3. after oxidation finishes, drip 0.25mLMPAA guanidine hydrochloride solution (0.2MMPAA, 6.0M Guanidinium hydrochloride, 0.2M phosphoric acid salt, pH 5.0), remove cryosel and bathe, get the acidity of the careful conditioned reaction liquid of NaOH (2.0M) solution to neutral (acidity is measured with the micro glass pH meter).
4. after reacting 2 hours, get the 20uL reaction solution and mixed 5 minutes with 60uL TCEP (30mM), HPLC detects, and separates the peak that makes new advances, and mass spectrum is defined as connecting product, and result as shown in figure 19.
Embodiment 20, the synthetic H-Leu-Tyr-Arg-Ser[β-D-Glc (OAc) of polypeptide hydrazides method
4]-Ala-Cys-Lys-Tyr-Met-His-OH
1. weigh polypeptide hydrazides H-Leu-Tyr-Arg-Ser[β-D-Glc (OAc)
4]-Ala-NHNH
2(2.0 milligrams), H-Cys-Lys-Tyr-Met-His-OH (1.95 milligrams) are dissolved in 0.65mLPBS (pH 3.0 for 6.0M Guanidinium hydrochloride, 0.2M phosphoric acid salt), add 50uL benzamide solution (40mM).
2. get the 0.25mL mentioned solution and put into the vial of 1.5mL, add magneton, (approximately-10 ℃ of left and right) stirs the lower 25uLNaNO of dropping in cryosel is bathed
2(0.2M) aqueous solution, low-temp reaction is about 20 minutes.
3. after oxidation finishes, drip 0.25mLMPAA guanidine hydrochloride solution (0.2MMPAA, 6.0M Guanidinium hydrochloride, 0.2M phosphoric acid salt, pH 5.0), remove cryosel and bathe, get the acidity of the careful conditioned reaction liquid of NaOH (2.0M) solution to neutral (acidity is measured with the micro glass pH meter).
4. after reacting 2 hours, get the 20uL reaction solution and mixed 5 minutes with 60uL TCEP (30mM), HPLC detects, and separates the peak that makes new advances, and mass spectrum is defined as connecting product, and result as shown in figure 20.
Embodiment 21, the synthetic Trifolitoxin[Ala23Cys of polypeptide hydrazides method]
1. weigh polypeptide hydrazides Trifolitoxin[Met
1-Ala
22]-NHNH
2(0.9 milligram), Trifolitoxin[Cys
23-Ala
42] (0.9 milligram) be dissolved in 0.12mLPBS (pH 3.0 for 6.0M Guanidinium hydrochloride, 0.2M phosphoric acid salt), adds 9uL benzamide solution (40mM).
2. mentioned solution is put into the vial of 1.5mL, adds magneton, and (approximately-10 ℃ of left and right) stirs the lower 12uLNaNO of dropping in cryosel is bathed
2(0.2M) aqueous solution, low-temp reaction is about 20 minutes.
3. after oxidation finishes, drip 0.12mLMPAA guanidine hydrochloride solution (0.2MMPAA, 6.0M Guanidinium hydrochloride, 0.2M phosphoric acid salt, pH 5.0), remove cryosel and bathe, get the acidity of the careful conditioned reaction liquid of NaOH (2.0M) solution to neutral (acidity is measured with the micro glass pH meter).
4. after reacting 2 hours, get the 20uL reaction solution and mixed 5 minutes with 60uL TCEP (30mM), HPLC detects, and separates the peak that makes new advances, and mass spectrum is defined as connecting product, and result as shown in figure 21.
Embodiment 22, the synthetic scorpion toxin bioactive peptide II (CssII) (result is referring to Figure 22,23 and 24) of polypeptide hydrazides method
1. weigh polypeptide hydrazides CssII[Lys
1-Gly
11]-NHNH
2(0.9 milligram) is dissolved in 0.25mLPBS (pH 3.0 for 6.0M Guanidinium hydrochloride, 0.2M phosphoric acid salt), adds 17uL benzamide solution (40mM).
2. mentioned solution is put into the vial of 1.5mL, adds magneton, and (about approximately-10oC) stirs the lower 23uLNaNO2 (0.2M) of the dropping aqueous solution in cryosel is bathed, and low-temp reaction is about 20 minutes.
3. after oxidation finishes, drip 0.12mLMPAA guanidine hydrochloride solution (0.2M phosphoric acid salt, pH 5.0 for 0.2MMPAA, 6.0M Guanidinium hydrochloride), remove cryosel and bathe, then add CssII[Cys
12-Tyr
24]-NHNH
2(1.2 milligrams) get the acidity of the careful conditioned reaction liquid of NaOH (2.0M) solution to neutral (acidity is measured with the micro glass pH meter).
4. after reacting 2 hours, get the 20uL reaction solution and mixed 5 minutes with 60uL TCEP (30mM), HPLC detects, and separates the peak that makes new advances, and mass spectrum is defined as connecting product C ssII[Lys
1-Tyr
24]-NHNH
2Adopt semipreparative column to isolate target product, the cryodesiccated the first step connects product, HPLC productive rate 92%, isolated yield 70%.
5. weigh polypeptide hydrazides CssII[Lys
1-Tyr
24]-NHNH
2(2.0 milligrams) are dissolved in 0.24mLPBS (pH 3.0 for 6.0M Guanidinium hydrochloride, 0.2M phosphoric acid salt), add 18uL benzamide solution (40mM).
6. mentioned solution is put into the vial of 1.5mL, adds magneton, and (about approximately-10oC) stirs the lower 24uLNaNO2 (0.2M) of the dropping aqueous solution in cryosel is bathed, and low-temp reaction is about 20 minutes.
7. after oxidation finishes, drip 0.12mLMPAA guanidine hydrochloride solution (0.2M phosphoric acid salt, pH 5.0 for 0.2MMPAA, 6.0M Guanidinium hydrochloride), remove cryosel and bathe, then add CssII[Cys
25-Tyr
40]-NHNH
2(4.0 milligrams) get the acidity of the careful conditioned reaction liquid of NaOH (2.0M) solution to neutral (acidity is measured with the micro glass pH meter).
8. after reacting 2 hours, get the 20uL reaction solution and mixed 5 minutes with 60uL TCEP (30mM), HPLC detects, and separates the peak that makes new advances, and mass spectrum is defined as connecting product C ssII[Lys
1-Tyr
40]-NHNH
2Adopt semipreparative column to isolate target product, cryodesiccated second step connects product, HPLC productive rate 85%, isolated yield 50%.
9. weigh polypeptide hydrazides CssII[Lys
1-Tyr
40]-NHNH
2(0.8 milligram) is dissolved in 0.12mLPBS (pH 3.0 for 6.0M Guanidinium hydrochloride, 0.2M phosphoric acid salt), adds 9uL benzamide solution (40mM).
10. mentioned solution is put into the vial of 1.5mL, adds magneton, and (about approximately-10oC) stirs the lower 6uLNaNO2 (0.2M) of the dropping aqueous solution in cryosel is bathed, and low-temp reaction is about 20 minutes.
11. after oxidation finishes, dropping 0.12mLMPAA guanidine hydrochloride solution (0.2MMPAA, the 6.0M Guanidinium hydrochloride, 0.2M phosphoric acid salt, pH 5.0), remove cryosel and bathe, then add CssII[Cys
41-Tyr
66]-NH
2(1.6 milligrams) get the acidity of the careful conditioned reaction liquid of NaOH (2.0M) solution to neutral (acidity is measured with the micro glass pH meter).
12. after reaction is spent the night, get the 20uL reaction solution and mixed 5 minutes with 60uL TCEP (30mM), HPLC detects, and separates the peak that makes new advances, mass spectrum is defined as connecting product C ssII, HPLC productive rate 50%.
Embodiment 23, the synthetic microtubule-associated protein light chain 3 (LC3) of polypeptide hydrazides method
1. design 5 ' the end primer (5 '-GGTGGTCATATGCCGTCCGAGAAGACCT-3 ') and 3 ' that contains respectively NdeI and BspQI restriction enzyme site is held primer (5 '-GGTGGTTGCTCTTCCGCACGTCTCCTGGGAGGC-3 '), and pcr amplification obtains LC3[Pro2-Thr118] DNA sequence dna.The pTXB1 coli expression carrier (New England Biolab, Beverly, MA) that cuts with enzyme in advance after DNA (QIAquick kit) after purifying amplification, NdeI and BspQI enzyme cut connects.The pTXB1 plasmid transfection that builds is advanced intestinal bacteria ER2566 (NEB) cell, be amplified to the growth of 1L nutrient solution, shake bacterium until OD600=0.6.Adding IPTG is 0.2mM to final concentration, and 16 ℃ of abduction deliverings spend the night.The ultrasonication cell, Eddy diffusion in cell pyrolysis liquid (20mM HEPES, pH 7.4,500mM NaCl, 0.1%Triton X-100 (v/v) and 1mM EDTA).The centrifuging and taking supernatant, after filtering membrane, loading is to using on the good chitin resin column of lysate balance in advance, and room temperature was cultivated 2 hours.Use the lysate washing resin post of 10 times of column volumes.Configuration contains the lysate of 8% hydrazine hydrate pH 7.4, obtains cutting liquid.Resin was cultivated in cutting liquid 1 to 2 hour, is obtained LC3[Pro2-Thr118 after wash-out]-NHNH2.Use again the lysate washing resin one time of 2 times of column volumes, merge elutriant, ultrafiltration and concentration, RP-HPLC collects the component postlyophilization, obtains 10mg hydrazides albumen.Through the ESI-MS mass spectral characteristi, obtain target product.
2. weigh polypeptide hydrazides LC3[Pro2-Thr118] 6.0mg, LC3[Cys119-Val125]-OH 0.6mg, be dissolved in 0.22mL and connect damping fluid (pH 3.0 for 6.0M Guanidinium hydrochloride, 0.2M phosphoric acid salt), add mark benzamide solution in 10uL.
3. mentioned solution is put into the Glass tubing of 1.5mL, adds magneton, and (about approximately-10oC) stirs the lower 22uLNaNO2 (0.2M) of the dropping aqueous solution in cryosel is bathed, and low-temp reaction is about 20 minutes.
4. after oxidation finishes, drip guanidine hydrochloride solution (0.2M MPAA, 6.0M Guanidinium hydrochloride that 0.12mL contains MPAA, 0.2M phosphoric acid salt, pH 5.0), remove cryosel and bathe, get the acidity of the careful conditioned reaction liquid of NaOH (2.0M) solution to neutral (acidity is measured with the micro glass pH meter).
5. after reacting 2 hours, get the 20uL reaction solution and mixed 5 minutes with 60uL TCEP (30mM), HPLC detects, and separates the peak that makes new advances, and mass spectrum is defined as connecting product, and result is referring to Figure 25.
Claims (6)
1. one kind prepares method of protein, and it comprises that the first peptide that through the following steps the C end is had the hydrazides group is connected for the second peptide of halfcystine with the N end:
Described the first peptide and described the second peptide are being contained NaNO
2Solution in mix and regulate pH to acid pH, obtain the first reaction mixture in order to the hydrazides group of described the first peptide is converted into the acid azide group;
Add in described the first reaction mixture and contain the solution of reductive agent and regulate pH to neutral pH; wherein said reductive agent contains mercapto groups; so that the acid azide groups converted of described the first peptide is thioester group; keep the C end thioesters of described neutral pH and then described the first peptide and the N end of described the second peptide ligation formation protein occurs
Wherein said acid pH is 3.0-4.0.
2. preparation method of protein according to claim 1, wherein said reductive agent is 4-sulfydryl phenylacetic acid.
3. preparation method of protein according to claim 1, wherein utilize sodium hydroxide to regulate pH to described neutral pH.
4. method for preparing the polypeptide thioesters compound is characterized in that comprising the following steps:
To have the first peptide of hydrazides group and contain NaNO as the C end of raw material
2Solution mix, and regulate pH to acid pH, obtain the first preparation feedback mixture in order to the hydrazides group of described the first peptide is converted into the acid azide group,
Add the solution that contains reductive agent in described the first preparation feedback mixture, and regulate pH to neutral pH, wherein said reductive agent contains mercapto groups, so that the acid azide groups converted of described the first peptide is thioester group, forms described polypeptide thioesters compound,
Wherein said acid pH is 3.0-4.0.
5. the method for preparing the polypeptide thioesters compound according to claim 4, wherein said reductive agent is 4-sulfydryl phenylacetic acid.
6. the method for preparing the polypeptide thioesters compound according to claim 4, wherein utilize sodium hydroxide to regulate pH to described neutral pH.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201110062686 CN102199214B (en) | 2011-03-15 | 2011-03-15 | Protein preparation method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201110062686 CN102199214B (en) | 2011-03-15 | 2011-03-15 | Protein preparation method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102199214A CN102199214A (en) | 2011-09-28 |
| CN102199214B true CN102199214B (en) | 2013-05-22 |
Family
ID=44660230
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 201110062686 Active CN102199214B (en) | 2011-03-15 | 2011-03-15 | Protein preparation method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102199214B (en) |
Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP6493216B2 (en) * | 2013-11-05 | 2019-04-03 | 味の素株式会社 | Process for producing peptide hydrazide, peptide amide and peptide thioester |
| CN103755843B (en) * | 2013-12-11 | 2016-06-01 | 清华大学 | N-9-fluorenylmethyloxycarbonyl hydrazine base resin and its preparation method and application |
| CN107935895B (en) * | 2017-11-22 | 2019-10-29 | 清华大学 | Compound and its preparation method and application |
| CN109111506A (en) * | 2018-01-17 | 2019-01-01 | 上海长征医院 | A kind of polypeptide and its preparation method and application for treating osteoporosis |
| CN111751468A (en) * | 2020-07-03 | 2020-10-09 | 上海艾魁英生物科技有限公司 | Method for purifying lysozyme dimer and preparing standard substance thereof |
| CN113185572B (en) * | 2021-05-24 | 2023-03-17 | 江南大学 | Extraction method of Pleurotus tuber-regium cell wall glycoprotein |
| CN114276430A (en) * | 2021-12-20 | 2022-04-05 | 上海普佑生物医药有限公司 | sDSS1 mutant protein synthesis method and purification process route thereof |
-
2011
- 2011-03-15 CN CN 201110062686 patent/CN102199214B/en active Active
Non-Patent Citations (7)
| Title |
|---|
| Chemoselective Ligation of Peptide Phenyl Esters with N-Terminal Cysteines;Ge-Min Fang et al.,;《ChemBioChem》;20100416;第11卷;1061-1065 * |
| Ge-Min Fang et al.,.Chemoselective Ligation of Peptide Phenyl Esters with N-Terminal Cysteines.《ChemBioChem》.2010,第11卷1061-1065. |
| Ge-Min Fang et al.,.Protein Chemical Synthesis by Ligation of Peptide Hydrazides.《Protein Chemical Synthesis》.2011,第50卷7645-7649. * |
| Ji-Shen Zheng et al.,.Chemical Protein Synthesis by Kinetically Controlled Ligation of Peptide O-Esters.《ChemBioChem》.2010,第11卷511-515. * |
| Ji-Shen Zheng et al.,.Fmoc Synthesis of Peptide Thioesters without Post-Chain-Assembly Manipulation.《American Chemical Society》.2011,第133卷11080-11083. * |
| 唐姗 等.多肽酰肼连接法合成环四肽.《化学学报》.2012,第70卷1471-1476. * |
| 方葛敏.面向蛋白质化学合成与修饰的新方法_新型多肽硫酯衍生物的研发和应用.《中国优秀硕士学位论文全文数据库工程科技I辑》.2010,(第7期),全文. * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102199214A (en) | 2011-09-28 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102199214B (en) | Protein preparation method | |
| Clonis | Affinity chromatography matures as bioinformatic and combinatorial tools develop | |
| US10239922B2 (en) | Fusion proteins of superfolder green fluorescent protein and use thereof | |
| Rocco et al. | Construction and use of new cloning vectors for the rapid isolation of recombinant proteins from Escherichia coli | |
| McGrath et al. | Chemoselectivity in chemical biology: acyl transfer reactions with sulfur and selenium | |
| FI113272B (en) | Improved protein folding method | |
| Nilsson et al. | Chemical synthesis of proteins | |
| ES2324951T3 (en) | PROCEDURE FOR THE PREPARATION OF CARBOXI-TERMINAL AMBED PEPTIDES. | |
| US5648244A (en) | Production, purification, cleavage and use of fusion peptides | |
| CA3051195A1 (en) | Split inteins with exceptional splicing activity | |
| CN109072203B (en) | Mirror image nucleic acid replication system | |
| WO2010102085A4 (en) | Stabilized reverse transcriptase fusion proteins | |
| US20090123972A1 (en) | Staphylococcal nuclease fusion proteins for the production of recombinant peptides | |
| WO2001098366A2 (en) | Enterokinase cleavage sequences and their use | |
| Shen et al. | Chemical synthesis of integral membrane proteins: methods and applications | |
| EP2550290A2 (en) | Method of modifying a specific lysine | |
| Tolbert et al. | Conjugation of glycopeptide thioesters to expressed protein fragments: semisynthesis of glycosylated interleukin-2 | |
| EP3904525A1 (en) | Fusion polypeptides for target peptide production | |
| Tam et al. | Tandem peptide ligation for synthetic and natural biologicals | |
| WO2015115661A1 (en) | Method for producing peptides having azole-derived skeleton | |
| Elmore | Peptide synthesis | |
| AU768443B2 (en) | Method for stabilising proteins in complex mixtures during their storage in aqueous solvents | |
| Gao et al. | Chemical synthesis of a 28 kDa full-length PET degrading enzyme ICCG by the removable backbone modification strategy | |
| Karskela et al. | Synthesis of bicyclic peptides | |
| EP4265636A1 (en) | Preparation of target peptides and proteins |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |