CN103601797A - Glycosylation modification method of hungtintin - Google Patents
Glycosylation modification method of hungtintin Download PDFInfo
- Publication number
- CN103601797A CN103601797A CN201310555234.8A CN201310555234A CN103601797A CN 103601797 A CN103601797 A CN 103601797A CN 201310555234 A CN201310555234 A CN 201310555234A CN 103601797 A CN103601797 A CN 103601797A
- Authority
- CN
- China
- Prior art keywords
- htt
- protein
- recombinant
- cys
- huntingtin
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 230000013595 glycosylation Effects 0.000 title claims abstract description 60
- 238000006206 glycosylation reaction Methods 0.000 title claims abstract description 59
- 238000002715 modification method Methods 0.000 title claims 2
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 81
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 72
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 44
- 229920001184 polypeptide Polymers 0.000 claims abstract description 41
- 102000004196 processed proteins & peptides Human genes 0.000 claims abstract description 41
- 238000000034 method Methods 0.000 claims abstract description 22
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 claims abstract description 20
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 claims abstract description 20
- 241000588724 Escherichia coli Species 0.000 claims abstract description 15
- 238000000746 purification Methods 0.000 claims abstract description 14
- 230000014509 gene expression Effects 0.000 claims abstract description 8
- 239000013613 expression plasmid Substances 0.000 claims abstract description 6
- 238000003259 recombinant expression Methods 0.000 claims abstract description 6
- 108010004034 stable plasma protein solution Proteins 0.000 claims abstract description 4
- 102000016252 Huntingtin Human genes 0.000 claims abstract 12
- 108050004784 Huntingtin Proteins 0.000 claims abstract 12
- 230000004048 modification Effects 0.000 claims abstract 8
- 238000012986 modification Methods 0.000 claims abstract 8
- 238000010276 construction Methods 0.000 claims abstract 6
- 239000012634 fragment Substances 0.000 claims description 29
- 239000013612 plasmid Substances 0.000 claims description 27
- 239000007788 liquid Substances 0.000 claims description 13
- 238000004458 analytical method Methods 0.000 claims description 9
- 239000011347 resin Substances 0.000 claims description 9
- 229920005989 resin Polymers 0.000 claims description 9
- 238000012216 screening Methods 0.000 claims description 7
- 241000894006 Bacteria Species 0.000 claims description 6
- 101150043003 Htt gene Proteins 0.000 claims description 6
- 230000008878 coupling Effects 0.000 claims description 5
- 238000010168 coupling process Methods 0.000 claims description 5
- 238000005859 coupling reaction Methods 0.000 claims description 5
- 239000007790 solid phase Substances 0.000 claims description 5
- 239000006228 supernatant Substances 0.000 claims description 5
- 230000008569 process Effects 0.000 claims description 4
- 108091008146 restriction endonucleases Proteins 0.000 claims description 4
- 238000012163 sequencing technique Methods 0.000 claims description 4
- 239000003875 Wang resin Substances 0.000 claims description 3
- NERFNHBZJXXFGY-UHFFFAOYSA-N [4-[(4-methylphenyl)methoxy]phenyl]methanol Chemical group C1=CC(C)=CC=C1COC1=CC=C(CO)C=C1 NERFNHBZJXXFGY-UHFFFAOYSA-N 0.000 claims description 3
- 239000002054 inoculum Substances 0.000 claims description 3
- 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 claims description 3
- 230000009465 prokaryotic expression Effects 0.000 claims description 3
- 239000000126 substance Substances 0.000 claims description 3
- 230000002194 synthesizing effect Effects 0.000 claims description 3
- PIEPQKCYPFFYMG-UHFFFAOYSA-N tris acetate Chemical compound CC(O)=O.OCC(N)(CO)CO PIEPQKCYPFFYMG-UHFFFAOYSA-N 0.000 claims description 3
- 238000011144 upstream manufacturing Methods 0.000 claims description 3
- 230000015572 biosynthetic process Effects 0.000 claims description 2
- 239000013599 cloning vector Substances 0.000 claims description 2
- 239000013604 expression vector Substances 0.000 claims description 2
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 claims 3
- 229960000723 ampicillin Drugs 0.000 claims 3
- 238000001976 enzyme digestion Methods 0.000 claims 2
- 238000012795 verification Methods 0.000 claims 2
- 241000620209 Escherichia coli DH5[alpha] Species 0.000 claims 1
- 238000005119 centrifugation Methods 0.000 claims 1
- 238000004520 electroporation Methods 0.000 claims 1
- 238000003786 synthesis reaction Methods 0.000 claims 1
- 239000013598 vector Substances 0.000 claims 1
- 238000010521 absorption reaction Methods 0.000 abstract description 12
- 102000035122 glycosylated proteins Human genes 0.000 description 14
- 108091005608 glycosylated proteins Proteins 0.000 description 14
- 238000001142 circular dichroism spectrum Methods 0.000 description 9
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 8
- 239000012071 phase Substances 0.000 description 7
- 239000000047 product Substances 0.000 description 7
- 239000000243 solution Substances 0.000 description 7
- DTQVDTLACAAQTR-UHFFFAOYSA-N trifluoroacetic acid Substances OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 7
- 108090000790 Enzymes Proteins 0.000 description 5
- 102000004190 Enzymes Human genes 0.000 description 5
- 208000023105 Huntington disease Diseases 0.000 description 5
- 210000004027 cell Anatomy 0.000 description 5
- 238000006243 chemical reaction Methods 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 4
- 239000007995 HEPES buffer Substances 0.000 description 4
- 238000011156 evaluation Methods 0.000 description 4
- 235000015097 nutrients Nutrition 0.000 description 4
- 239000011780 sodium chloride Substances 0.000 description 4
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 3
- PMZXXNPJQYDFJX-UHFFFAOYSA-N acetonitrile;2,2,2-trifluoroacetic acid Chemical compound CC#N.OC(=O)C(F)(F)F PMZXXNPJQYDFJX-UHFFFAOYSA-N 0.000 description 3
- 150000001413 amino acids Chemical class 0.000 description 3
- 239000007864 aqueous solution Substances 0.000 description 3
- 238000002983 circular dichroism Methods 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 230000029087 digestion Effects 0.000 description 3
- 239000000284 extract Substances 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 125000003088 (fluoren-9-ylmethoxy)carbonyl group Chemical group 0.000 description 2
- OGYGFUAIIOPWQD-UHFFFAOYSA-N 1,3-thiazolidine Chemical compound C1CSCN1 OGYGFUAIIOPWQD-UHFFFAOYSA-N 0.000 description 2
- 229920002101 Chitin Polymers 0.000 description 2
- 206010012289 Dementia Diseases 0.000 description 2
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 2
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 description 2
- -1 azoles alkane Chemical class 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 238000013016 damping Methods 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000004090 dissolution Methods 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- 230000004927 fusion Effects 0.000 description 2
- 230000007062 hydrolysis Effects 0.000 description 2
- 238000006460 hydrolysis reaction Methods 0.000 description 2
- XNXVOSBNFZWHBV-UHFFFAOYSA-N hydron;o-methylhydroxylamine;chloride Chemical compound Cl.CON XNXVOSBNFZWHBV-UHFFFAOYSA-N 0.000 description 2
- 238000011081 inoculation Methods 0.000 description 2
- 230000017730 intein-mediated protein splicing Effects 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 230000009145 protein modification Effects 0.000 description 2
- 238000004366 reverse phase liquid chromatography Methods 0.000 description 2
- 238000004007 reversed phase HPLC Methods 0.000 description 2
- 238000002525 ultrasonication Methods 0.000 description 2
- 239000003643 water by type Substances 0.000 description 2
- 239000005059 1,4-Cyclohexyldiisocyanate Substances 0.000 description 1
- KKTUQAYCCLMNOA-UHFFFAOYSA-N 2,3-diaminobenzoic acid Chemical compound NC1=CC=CC(C(O)=O)=C1N KKTUQAYCCLMNOA-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
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- JRQLZCFSWYQHPI-UHFFFAOYSA-N 4,5-dichloro-2-cyclohexyl-1,2-thiazol-3-one Chemical compound O=C1C(Cl)=C(Cl)SN1C1CCCCC1 JRQLZCFSWYQHPI-UHFFFAOYSA-N 0.000 description 1
- 206010010947 Coordination abnormal Diseases 0.000 description 1
- 101000925662 Enterobacteria phage PRD1 Endolysin Proteins 0.000 description 1
- 241000206602 Eukaryota Species 0.000 description 1
- 102000003886 Glycoproteins Human genes 0.000 description 1
- 108090000288 Glycoproteins Proteins 0.000 description 1
- 102400000597 Glycosylated protein A Human genes 0.000 description 1
- 101800002131 Glycosylated protein A Proteins 0.000 description 1
- XOGTZOOQQBDUSI-UHFFFAOYSA-M Mesna Chemical compound [Na+].[O-]S(=O)(=O)CCS XOGTZOOQQBDUSI-UHFFFAOYSA-M 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 101000702488 Rattus norvegicus High affinity cationic amino acid transporter 1 Proteins 0.000 description 1
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 1
- 108060008539 Transglutaminase Proteins 0.000 description 1
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 1
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 150000001408 amides Chemical group 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 208000021018 autosomal dominant inheritance Diseases 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 210000004958 brain cell Anatomy 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 238000001311 chemical methods and process Methods 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 230000008034 disappearance Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 1
- 125000000350 glycoloyl group Chemical group O=C([*])C([H])([H])O[H] 0.000 description 1
- 125000003147 glycosyl group Chemical group 0.000 description 1
- 230000005484 gravity Effects 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000000816 matrix-assisted laser desorption--ionisation Methods 0.000 description 1
- 238000001840 matrix-assisted laser desorption--ionisation time-of-flight mass spectrometry Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 229960004635 mesna Drugs 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 210000005036 nerve Anatomy 0.000 description 1
- 230000001537 neural effect Effects 0.000 description 1
- 230000004770 neurodegeneration Effects 0.000 description 1
- 208000015122 neurodegenerative disease Diseases 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 230000026731 phosphorylation Effects 0.000 description 1
- 238000006366 phosphorylation reaction Methods 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 230000000750 progressive effect Effects 0.000 description 1
- 239000012460 protein solution Substances 0.000 description 1
- 208000020016 psychiatric disease Diseases 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000010532 solid phase synthesis reaction Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 150000007970 thio esters Chemical class 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 102000003601 transglutaminase Human genes 0.000 description 1
- 230000034512 ubiquitination Effects 0.000 description 1
- 238000010798 ubiquitination Methods 0.000 description 1
- 238000004704 ultra performance liquid chromatography Methods 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
- 230000003313 weakening effect Effects 0.000 description 1
Images
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
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
Landscapes
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Engineering & Computer Science (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- General Health & Medical Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Microbiology (AREA)
- Plant Pathology (AREA)
- Physics & Mathematics (AREA)
- Toxicology (AREA)
- Gastroenterology & Hepatology (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Peptides Or Proteins (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
本发明公开了一种亨廷顿蛋白的糖基化修饰方法，包括以下步骤：（1）获取重组蛋白Htt（1-90）：a.亨廷顿蛋白基因的获得；b.重组表达质粒pTWIN1-htt的构建；c.表达亨廷顿蛋白的重组大肠杆菌菌株DH5α-htt的构建；d.重组蛋白的表达；（2）重组蛋白Htt（1-90）的纯化；（3）SPPS方法获得糖基化修饰的Htt（Cys-K92-K158）多肽；（4）Htt（Cys-K92-K158）多肽的纯化；（5）Htt（Cys-K92-K158）多肽与重组蛋白Htt（1-90）经过偶联获得经糖基化修饰目的蛋白Htt（1-158）；（6）经糖基化修饰目的蛋白Htt（1-158）的纯化。通过对亨廷顿蛋白的糖基化修饰，显示出糖基化蛋白有较强的稳定性，且温度和时间对亨廷顿蛋白的吸收峰的影响，说明经糖基化修饰的亨廷顿蛋白相对稳定。
The invention discloses a method for glycosylation modification of huntingtin protein, comprising the following steps: (1) Obtaining recombinant protein Htt (1-90): a. Obtaining huntingtin gene; b. Construction of recombinant expression plasmid pTWIN1- htt ; c. Construction of recombinant E. coli strain DH5α-htt expressing huntingtin protein; d. Expression of recombinant protein; (2) Purification of recombinant protein Htt (1-90); (3) SPPS method to obtain glycosylated modified Htt (Cys-K92-K158) polypeptide; (4) Purification of Htt (Cys-K92-K158) polypeptide; (5) Htt (Cys-K92-K158) polypeptide and recombinant protein Htt (1-90) were coupled to obtain the Glycosylation modified target protein Htt (1-158); (6) Purification of glycosylated target protein Htt (1-158). Glycosylation modification of huntingtin protein shows that glycosylation protein has strong stability, and the influence of temperature and time on the absorption peak of huntingtin protein shows that glycosylation modified huntingtin protein is relatively stable.
Description
Technical field
The present invention relates to a kind of chemically modified of protein, be specifically related to a kind of glycosylation modified method of Huntington protein.
Background technology
Protein modification is a complicated process, and in eukaryote, modified types is a lot, common are glycosylation, acetylize, ubiquitination, phosphorylation and SUMOization, and protein modification can change activity, location or the function of protein.Protein glycosylation has important impact to the function of protein and stability, first many misfolded proteins, cellular activity and tissue metabolism are produced to certain influence, and then have influence on the function of organ, the type of attachment of glycosylated protein mainly contains N, O and connects two kinds of forms.Circular dichroism spectrum is absorbed in the change of protein structure and resistant to hydrolysis enzyme ability has a certain impact; Secondly, some glycosylated proteins are conducive to the correct folding of protein.
Huntington Chorea (Huntington disease, HD) be a kind of neurodegenerative disease of autosomal dominant inheritance, Huntington protein (the Huntingtin that one of HD genes encoding is comprised of 3144 amino acid, Htt), from the 17th of Htt amino-terminal end, start to have glutamine (CAG) sequence of one section of repetition.CAG tumor-necrosis factor glycoproteins will produce two direct results: the one, make wild-type Huntington protein (wild huntingtin, wHtt) disappearance, and the 2nd, produce saltant type Huntington protein (mutant Htt, mHtt).Excessive prolongation along with poly glumine, Htt albumen can occur to be cross-linked and formation aggregate under the effect of trans-glutaminases, under the effect connecting at hydrogen bond between main chain and amide side chain base, poly glumine sequence can form polymer, is called poly glumine sequence (PolyQ).Normal people's PolyQ length is less than 35, and HD patient's PolyQ length can be over 37.These abnormal proteins are accumulated into piece, broken parts brain cell, and particularly those control relevant cell with muscle, cause patient's neural system to be degenerated gradually, nerve impulse disperse, asynergia, there is uncontrollable ballism, and can develop into dementia, even dead.Clinical manifestation is mainly dancing sample involuntary action, mental disorder and progressive dementia.
Do not have the Huntington protein of modifying to demonstrate less stable etc., at present domestic also do not have institute or mechanism to carry out chemically modified to Huntington protein.Glycosylation modified by Huntington protein, the O-that is mainly Ser and Thr is connected glycosylation, the stability that demonstrates albumen increases, glycosylated protein has different circular dichroism spectrums to absorb, demonstrate glycosylated protein and have stronger stability, and the impact of temperature and time on the absorption peak of Huntington protein, illustrates through glycosylation modified Huntington protein relatively stablely, and the glycosylation of therefore studying Huntington protein is significant to the treatment of huntington disease.
Summary of the invention
For the deficiencies in the prior art, a kind of glycosylation modified method that the object of this invention is to provide Huntington protein, glycosylation modified by Huntington protein, the O-that is mainly Ser and Thr is connected glycosylation, the stability that demonstrates albumen increases, glycosylated protein has different circular dichroism spectrums to absorb, demonstrate glycosylated protein and have stronger stability, and the impact of temperature and time on the absorption peak of Huntington protein, explanation is relatively stable through glycosylation modified Huntington protein, therefore the glycosylation of studying Huntington protein is significant to the treatment of huntington disease.
Object of the present invention can realize by following technical proposal: a kind of glycosylation modified method of Huntington protein, comprises the following steps: (1), obtain recombinant protein Htt(1-90): the acquisition of a, Huntington protein gene; B, recombinant expression plasmid pTWIN1-
httstructure; The structure of the recombinant escherichia coli strain DH5 α-htt of c, expression Huntington protein; The expression of d, recombinant protein; (2) purifying, recombinant protein Htt(1-90); (3), SPPS method obtains glycosylation modified Htt(Cys-K92-K158) polypeptide; (4) Htt(Cys-K92-K158) purifying of polypeptide; (5) Htt(Cys-K92-K158) polypeptide and recombinant protein Htt(1-90) through coupling, obtain through glycosylation modified target protein Htt(1-158); (6) through glycosylation modified target protein Htt(1-158) purifying.
As preferably, in described step (1): the acquisition of a, Huntington protein gene: design contains
ecor and
pstupstream and downstream primer P1 and the P2 of restriction site, from the method amplification Huntington protein gene fragment of plasmid pcDNA3.1/mys-His employing PCR, by increased
httgene fragment and the link of cloning vector pMD18-T carrier, obtain recombinant plasmid, proceeds in e. coli jm109, through amicillin resistance screening, picking positive colony, extracts plasmid and carries out enzyme and cut evaluation and sequencing analysis checking, gained recombinant plasmid called after pMD18T-
htt; The structure of b, recombinant expression plasmid pTWIN1-htt: by prokaryotic expression carrier pTWIN1 and recombinant plasmid pMD18T-
httuse respectively restriction enzyme
ecor and
pstdouble digestion, after purification process by linear pTWIN1 plasmid fragment and
httgene fragment connects, and in conversion e. coli jm109, through amicillin resistance screening, picking positive colony, extracts plasmid and carry out enzyme and cut evaluation and sequencing analysis checking, gained recombinant plasmid called after pTWIN1-
htt; The structure of the recombinant escherichia coli strain of c, expression Huntington protein: adopt electrotransformation by recombinant plasmid pTWIN1-
httbe transformed in bacillus coli DH 5 alpha competent cell, utilize amicillin resistance screening, picking positive colony, obtains recombinant bacterium, called after: DH5 α-
htt; The expression of d, recombinant protein: by recombinant bacterium DH5 α-
httbe seeded to LB liquid nutrient medium, put 37 ℃ of shaking table incubated overnight, draw incubated overnight thalline with 5% inoculum size inoculation LB liquid nutrient medium, 37 ℃ of shaking tables are cultured to OD600=0.5 ~ 0.7, then put 20 ℃ of shaking tables and cultivate the IPTG abduction delivering target protein 4 ~ 6h that adds 1mmol/L, centrifugal collection thalline, at the TRIS-Acetate of 40mmol/L, carry out ultrasonication in the solution of 5mmol/L pH=8.3, by centrifugal (23000 * g, 30min, 40 ℃), cell debris is separated with supernatant liquor.
Further, synthetic Htt(Cys-K92-K158 in described step (3)) process of polypeptide is divided into three segment condense: a, first synthetic Cys-S138-K158 fragment; B, resynthesis Cys-E110-L136 fragment; C, last synthetic Cys-K92-I108 fragment; D, each fragment are by chemistry connection NCL or the connection of EPL method obtain Htt(Cys-K92-K158 naturally) polypeptide.
Further, glycosylation modified at least one site that is arranged in huntington exon 2 or ，JiS95 site, exon 3 region, T97 site, T107 site, S116 site, S120 site, S138 site, S143 site in described step (3).
Further, synthetic Cys-K92-I108 fragment solid phase carrier used is wang-resin.
Further, synthetic Cys-E110-L136 fragment and Cys-S138-K158 fragment solid phase carrier used are N-acyl group-benzoglyoxaline ketone resins.
Through glycosylation modified Huntington protein and the comparison of wild-type Huntington protein, have the following advantages: glycosylation modified by Huntington protein, the O-that is mainly Ser and Thr is connected glycosylation, the stability that demonstrates albumen increases, glycosylated protein has different circular dichroism spectrums to absorb, demonstrate glycosylated protein and have stronger stability, and the impact of temperature and time on the absorption peak of Huntington protein, explanation is relatively stable through glycosylation modified Huntington protein, and the glycosylation of therefore studying Huntington protein is significant to the treatment of huntington disease.
Accompanying drawing explanation
Fig. 1 is for obtaining the schema of Htt1-90 recombinant protein;
Fig. 2 is the glycosylation modified Htt(Cys-K92-K158 in S95 site) the synthetic schematic diagram of polypeptide;
Fig. 3 is through glycosylation modified target protein Htt(1-158) synthetic schemes;
Fig. 4: A is HttCys-K92-K158 protein sequence and synthetic fragment;
B is Htt(Cys-K92-K158) the glycosylation modified site of polypeptide;
Fig. 5: be the circular dichroism spectrum comparison of glycosylation and non-glycosylated Huntington protein:
A is the circular dichroism spectrogram of glycosylation Huntington protein;
B is the circular dichroism spectrogram of non-glycosylated Huntington protein;
Fig. 6: be the comparison of time to glycosylation Huntington protein and non-glycosylated Huntington protein stability influence:
A is that the time is to the stability analysis of glycosylation Huntington protein;
B is that the time is to non-glycosylated Huntington protein stability analysis;
Fig. 7: be the comparison of temperature to glycosylation Huntington protein and non-glycosylated Huntington protein stability influence:
A is that temperature is to the stability analysis of glycosylation Huntington protein;
B is that temperature is to non-glycosylated Huntington protein stability analysis.
Specific embodiment
embodiment 1:obtain recombinant protein Htt1-90
One) test materials
(1) carrier and bacterial strain
Plasmid pcDNA3.1/mys-His is so kind as to give by CHDI; Expression vector pTWIN1 is purchased from NEB company; Plasmid pMD18-T and e. coli jm109, DH5 α are purchased from Takara bio tech ltd.
(2) primer
P1：5-CCG
GAATTCCTGCCGTGCC-3 (
EcoR )
P2：5-AAAA
CTGCAGACAGCCGGGC-3 (
Pst )
Synthetic by Shanghai Sheng Gong biotechnology company limited.
Two) embodiment
1. the acquisition of Huntington protein gene
Take plasmid pcDNA3.1/mys-His as template, and design is synthetic to be contained
ecor and
pstupstream and downstream primer P1 and the P2 of restriction site, pcr amplification
httgene fragment, PCR product reclaims after agarose gel electrophoresis detects, and after purification process, is connected with carrier pMD18-T, proceeds in e. coli jm109, by amicillin resistance, screens, and obtains recombinant plasmid pMD18T-
htt, deliver to the order-checking of Shanghai Sheng Gong biotechnology company limited.Recombinant plasmid after checking is used
ecor and
pstdouble digestion is processed, and enzyme is cut product purification and processed rearmounted 4 ℃ of Refrigerator stores.
2. recombinant expression plasmid pTWIN1-
httstructure
By prokaryotic expression carrier pTWIN1 restriction enzyme
ecor and
pstdouble digestion, after purification process by linear pTWIN1 plasmid fragment and
httgene fragment connects, and transforms in e. coli jm109, and through amicillin resistance screening, picking positive colony, extracts plasmid and carry out enzyme and cut evaluation, obtains recombinant plasmid pTWIN1-
htt.
3. express the structure of the recombinant escherichia coli strain of Huntington protein
Adopt electrotransformation by recombinant plasmid pTWIN1-
httbe transformed in bacillus coli DH 5 alpha competent cell, utilize amicillin resistance screening, picking positive colony, obtain recombinant bacterium DH5 α-
htt.
By recombinant bacterium DH5 α-
httbe seeded to LB liquid nutrient medium, put 37 ℃ of shaking table incubated overnight.Draw incubated overnight thalline with 5% inoculum size inoculation LB liquid nutrient medium, 37 ℃ of shaking tables are cultured to OD600=0.5 ~ 0.7, then put 20 ℃ of shaking tables and cultivate the IPTG abduction delivering target protein 4 ~ 6h that adds 1mmol/L, centrifugal collection thalline.At the TRIS-Acetate of 40mmol/L, carry out ultrasonication in the solution of 5mmol/L pH=8.3.By centrifugal (23000 * g, 30min, 40 ℃), cell debris and supernatant liquor are separated.Thick polypeptide purity is 60%.Be illustrated in figure 1 and obtain recombinant protein Htt(1-90) schema.
embodiment 2:recombinant protein Htt(1-90) purifying
In Htt1-90-intein-CBD fusion rotein CBD composition can with chitin resin specific combination, thereby be convenient to remove foreign protein, subsequently intein composition under certain condition catalysis fusion rotein between htt1-90 and intein, rupture.Concrete operations are as follows: supernatant liquor is splined on to 2ml chitin gravity post.Pillar first passes through A liquid (Na-HEPES(pH8.0) 20mmol/L, NaCl 500mmol/L, EDTA 1mmol/L) carry out balance, after supernatant liquor loading, then with A liquid wash-out.Then pillar is at B liquid (Na-HEPES(pH8.0) 20mmol/L, NaCl 500mmol/L, EDTA 1mmol/L, DTT 50mmol/L) in soak 16h under 40C.C liquid (Na-HEPES(pH8.0) 20mmol/L, NaCl 500mmol/L) eluted protein, every 1ml collects.The sample that every step is collected carries out SDS-PAGE analysis, and the purity of identification of protein is 90%.
In eluted protein solution, add the solution that contains 0.25mmol/L pH7.8 MESNA, room temperature shaking table cutting 3 times, each 24h.Collect the HEPES Buffer slow rinse pillar that cutting liquid adds 1 times of volume, merge secondary elutriant, last cutting liquid pressurization dries up and reclaims resin.Albumen elutriant is moved to super filter tube, the centrifugal 15-30min of 10000r/min, ultrafiltration during to 1.5ml liquid-transfering gun shift upper strata protein liquid, obtain purity and be 99% recombinant polypeptide 2g ,-40 ℃ frozen.
embodiment 3: SPPS method obtains and is positioned at the glycosylation modified Htt(Cys-K92-K158 in S95 site) polypeptide
This building-up process is divided into three fragments, first synthetic Cys-S138-K158, resynthesis Cys-E110-L136, finally synthetic Cys-K92-I108.Each fragment is by chemistry connection NCL or the connection of EPL method obtain Htt(Cys-K92-K158 naturally) polypeptide
The synthetic of solid-phase peptide is to carry out on the Peptide synthesizer of CS336X, and the synthetic of Cys-S138-K158 polypeptide is based on Wang resin.Go protection and the depolymerization of resin can spontaneously be carried out in (TFA/DCM/H2O/TIPS 90/5/2.5/2.5) solution.Then the cold diethyl ether by 10 times of equivalents precipitates it, and these original polypeptide carry out purifying by reversed-phase HPLC, uses waters 600 pumps and waters 2489 ultraviolet/visible light detectors, and pillar adopts GRACE-Vydac 218TP54 C18 post.Mobile phase A is 0.1% trifluoroacetic acid aqueous solution, and Mobile phase B is 0.1% trifluoroacetic acid acetonitrile solution, and the detection wavelength of polypeptide is 214nm and 234nm.The quality of polypeptide is analyzed by MALDI-TOF-MS and ESI-MSF.The Cys-S138-K158 polypeptide that finally to obtain 16mg, purity be 99%, productive rate is 45%.
Cys-E110-L136 polypeptide is synthetic based on NBZ resin, produces a NBZ derived structure conveniently carry out NCL reaction at the C of polypeptide end.The initial preparation of NBZ resin is that 4 diaminobenzoic acid couplings obtain by the Di-Fmoc-3 of the mbha resin of amino acidifying and de-protected Fmoc and 5 times of equivalents.The amino acid that the prolongation of polypeptide is protected by Fmoc when DIPEA concentration reaches 0.5mol/L, forms ring-type in molecule when Dbz and the coupling of 4-chloroformate nitrophenyl ester.The polypeptide that finally fracture by key obtains, synthetic polypeptide N end adopts the protection of match azoles alkane, and under methoxyamine HCl pH4.0 condition, thiazolidine chemical bond rupture forms sulfydryl.Purification step is the same.The Cys-E110-L136 polypeptide that finally to obtain 14mg, purity be 99%, productive rate is 52%.
Cys-K92-I108 is synthetic synthetic similar with E110-L136 polypeptide, and the solid-state of polypeptide synthesized based on NBZ resin, produces a NBZ derived structure conveniently carry out NCL reaction at the C of polypeptide end.First by the glycoloyl on glycosyl, by the Serine hydroxyl generation chemical reaction of chemical process and S95 position, and then react with the L94 amino acids NCL of amino Fmoc protection.By the synthetic K92-I108-GlycoS95 polypeptide that obtains of solid peptide.Finally synthetic polypeptide is at N end match azoles alkane protection and C end NBZ derived structure, and under methoxyamine HCl pH4.0 condition, thiazolidine chemical bond rupture forms sulfydryl, and purification step is the same.The Cys-E110-L136 polypeptide that finally to obtain 10mg, purity be 99%, productive rate is 54%.
Be illustrated in figure 2 the glycosylation modified Htt(Cys-K92-K158 in S95 site) the synthetic schematic diagram of polypeptide.
embodiment 4: be positioned at S95 site through glycosylation modified Htt(Cys-K92-K158) purifying of polypeptide
The evaluation of polypeptide and purifying are by refilling reverse-phase chromatography, and now detector is diode-array detector, and moving phase is same.Mobile phase A is 0.1% trifluoroacetic acid aqueous solution, and Mobile phase B is 0.1% trifluoroacetic acid acetonitrile solution, and the detection wavelength of polypeptide is 214nm and 234nm.Finally obtain the HttCys-K92-K158 albumen of 5.04mg, productive rate is 12.6%.
embodiment 5:htt(Cys-K92-K158) polypeptide and recombinant protein Htt(1-90) through coupling, obtain through glycosylation modified target protein Htt(1-158)
Recombinant protein Htt1-90 and solid phase synthesis albumen HttCys-K92-K158 are put into damping fluid, utilize the specific reaction of sulfydryl and thioesters, then through completing synthesizing of peptide bond by S to the transformation of N.Its synthetic schematic diagram is as Fig. 3.
embodiment 6:through glycosylation modified target protein Htt(1-158) purifying
The reacted Htt1-158 product of NCL is by RPLC purifying, purification column is COSMOSIL 5C4-AR-300 (4.6mm*150mm 5 μ m), linear gradient is 10-90%, product wash-out is under 55% gradient, obtain product 4.5mg (0.5 μ mol, 33%), by MALDI, calculate the molecular weight of product.By UPLC, evaluate the purity of product.Purity is 99%, and productive rate is 6.3%.
embodiment 7:circular dichroism spectrum is analyzed the stability of glycosylation and non-glycosylated protein
The preparation of sample: sample dissolution at the tris-HCl of 10mmol/L, in the buffered soln of the NaCl of 75mmol/L, pH7.4, under 250C, with Jusco J185 circular dichroism spectrometer, analyze, within the scope of 190-250nm, average every 8-12nm collects wavelength, passing with 50nm/min speed of data scans, and every 0.2nm obtains a circular dichroism spectrum absorption value.And then the different circular dichroism spectrums that obtain two kinds of protein absorb.
Be illustrated in figure 5 the circular dichroism spectrum comparison of glycosylation and non-glycosylated Huntington protein, glycosylation modified huntington and wild-type Huntington protein are all to occur minimum absorption peak at 203nm, show Huntington protein and there is random coil structure, at 37 ℃, through cultivation after a while, glycosylation modified albumen absorption peak signal weakening, wild-type weakened 53%, through glycosylation modified glycoprotein, weakened 45%, shown that water-soluble protein produces cohesion and precipitation.Wild-type protein stability than glycosylated protein a little less than.
embodiment 8:glycosylated protein and the comparison of non-glycosylated Huntington protein stability
Sample preparation: sample dissolution, in damping fluid, by reverse-phase chromatographic column, is analyzed.
Chromatographic condition: by anti-phase-high performance liquid chromatography (RP-HPLC), waters2795 system, C18 post (4.6mm*250mm, 5 μ m).Mobile phase A: 0.1% trifluoroacetic acid aqueous solution, B is 0.1% trifluoroacetic acid acetonitrile solution.
Experimental result is as shown in the figure: in Fig. 6, non-glycosylated protein probably goes out peak 13min is all, along with time lengthening, its protein mass reduces, non-glycosylated protein under the effect of lytic enzyme, partial hydrolysis, after 1d, absorption peak has obvious reduction, and especially, after 3d, absorption peak is 25% of the zero hour.Be mainly to condense because glycosylated protein is not unstable, absorption peak is reduced.
In Fig. 7, glycosylation Huntington protein goes out peak in 13min left and right, illustrates that glycosylation has produced impact to appearance time, and at different temperature, its absorption peak has certain difference, and under hot conditions, absorption peak reduces, especially non-glycosylated protein.
Presentation of results, relatively stable through glycosylation modified Huntington protein in sum.
Claims (6)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310555234.8A CN103601797B (en) | 2013-11-11 | 2013-11-11 | The glycosylation modified method of Huntington protein |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310555234.8A CN103601797B (en) | 2013-11-11 | 2013-11-11 | The glycosylation modified method of Huntington protein |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103601797A true CN103601797A (en) | 2014-02-26 |
| CN103601797B CN103601797B (en) | 2015-09-30 |
Family
ID=50120073
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310555234.8A Active CN103601797B (en) | 2013-11-11 | 2013-11-11 | The glycosylation modified method of Huntington protein |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103601797B (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2424577A1 (en) * | 2002-04-22 | 2003-10-22 | The University Of British Columbia | Use of huntingtin and related compounds for modulating cell survival |
| WO2007082909A2 (en) * | 2006-01-18 | 2007-07-26 | Institut Curie | Method for treating huntington's disease by inhibiting dephosphorylation of huntingtin at s421 |
| US20110039789A1 (en) * | 2009-08-14 | 2011-02-17 | Institut Curie | Use of Huntingtin Protein for the Diagnosis and the Treatment of Cancer |
-
2013
- 2013-11-11 CN CN201310555234.8A patent/CN103601797B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2424577A1 (en) * | 2002-04-22 | 2003-10-22 | The University Of British Columbia | Use of huntingtin and related compounds for modulating cell survival |
| WO2007082909A2 (en) * | 2006-01-18 | 2007-07-26 | Institut Curie | Method for treating huntington's disease by inhibiting dephosphorylation of huntingtin at s421 |
| US20110039789A1 (en) * | 2009-08-14 | 2011-02-17 | Institut Curie | Use of Huntingtin Protein for the Diagnosis and the Treatment of Cancer |
Non-Patent Citations (3)
| Title |
|---|
| HARRISON,L.M.等: "NP_002102.4", 《NCBI》 * |
| YAN WANG等: "The role of post-translational modifications of huntingtin in the pathogenesis of Huntington’s disease", 《NEUROSCI BULL》 * |
| 王爱娥等: "一种亨廷顿舞蹈症体外药物筛选细胞模型的建立", 《中国生物工程杂志》 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103601797B (en) | 2015-09-30 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| KR102135976B1 (en) | Peptide with collagen synthesis-promoting activity and methods using the same | |
| CN110029118B (en) | A kind of method for synthesizing quercetin-4'-glucoside | |
| CN102839182B (en) | Method for preparing recombinant human nerve growth factor by using Escherichia coli expression system | |
| CN111117977B (en) | Recombinant polypeptide linked zymogen, preparation method, activation method and application thereof | |
| CN104611313A (en) | Beta-glucosidase as well as preparation method and application thereof | |
| CN101144093B (en) | Recombination expression carrier and method for soluble expressing human I-type metallothionin | |
| CN109321549A (en) | A kind of directional modification enzyme of heparinase I with improved specific enzyme activity, molecular modification method and expression engineering bacteria | |
| CN118496317A (en) | Lentinus edodes stem protein source DPP-IV and ACE dual-inhibitory peptide EW-5 and application thereof | |
| CN103103173B (en) | Chondrosulphatase B fusion protein, and coding gene and construction method thereof | |
| CN101608178A (en) | Fusion heparanase and its coding gene and preparation method | |
| CN103710367B (en) | A kind of recombined human kallikrein 1 and encoding gene thereof and preparation method | |
| WO2022253266A1 (en) | Recombinant protein purification method | |
| CN103601797A (en) | Glycosylation modification method of hungtintin | |
| KR101015343B1 (en) | Arabinos isomerase variants with enhanced enzyme activity | |
| CN109384852A (en) | Recombinate preparation, characterization and the application of Martentoxin | |
| CN102121013A (en) | Mature chicken interferon-alpha gene capable of high-efficiency expression and preparation method of polypeptide thereof | |
| CN101892253A (en) | Method for preparing thrombolytic drug reteplase without inclusion body renaturation in Escherichia coli | |
| CN103601798B (en) | The acylations modifying method of Huntington protein | |
| CN108823230A (en) | A kind of Prokaryotic expression, purification method of source of people selenoprotein H | |
| CN103601799A (en) | Phosphorylation modification method of hungtintin | |
| RU2453604C1 (en) | Hybrid protein (versions), bacterial strain escherichia coli - hybrid protein producer (versions) and method for producing methionine-free human interferon alpha-2 | |
| CN101139592A (en) | New Signal Cono O-Superfamily Toxin Sequence, Preparation Method and Application | |
| CN105209480A (en) | Protransduzin b, a gene transfer enhancer | |
| CN103601796A (en) | Palmitoylation modification method of hungtintin | |
| CN108165567A (en) | The synthesis of human lysozyme gene and its construction method of expression product |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20200513 Address after: No.514, building 1, No.1, Wufu Road, tianzihu Industrial Park, Anji County, Huzhou City, Zhejiang Province Patentee after: Zhejiang Puti Biotechnology Co.,Ltd. Address before: Yuhang District, Hangzhou City, Zhejiang province 311121 Wenyi Road No. 1500 Building No. 6 Patentee before: BSAZ BIOTECH (HANGZHOU) Co.,Ltd. |
|
| TR01 | Transfer of patent right | ||
| PE01 | Entry into force of the registration of the contract for pledge of patent right | ||
| PE01 | Entry into force of the registration of the contract for pledge of patent right |
Denomination of invention: Glycosylation Modification of Huntington Protein Effective date of registration: 20221129 Granted publication date: 20150930 Pledgee: Zhejiang Anji Rural Commercial Bank Co.,Ltd. Changhe District Sub branch Pledgor: Zhejiang Puti Biotechnology Co.,Ltd. Registration number: Y2022330003299 |
|
| PC01 | Cancellation of the registration of the contract for pledge of patent right | ||
| PC01 | Cancellation of the registration of the contract for pledge of patent right |
Granted publication date: 20150930 Pledgee: Zhejiang Anji Rural Commercial Bank Co.,Ltd. Changhe District Sub branch Pledgor: Zhejiang Puti Biotechnology Co.,Ltd. Registration number: Y2022330003299 |
|
| PE01 | Entry into force of the registration of the contract for pledge of patent right | ||
| PE01 | Entry into force of the registration of the contract for pledge of patent right |
Denomination of invention: Glycosylation modification methods of Huntington protein Granted publication date: 20150930 Pledgee: Zhejiang Anji Rural Commercial Bank Co.,Ltd. Changhe District Sub branch Pledgor: Zhejiang Puti Biotechnology Co.,Ltd. Registration number: Y2024330002457 |