CN103601797A - Glycosylation modification method of hungtintin - Google Patents
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Abstract
The invention discloses a glycosylation modification method of hungtintin. The glycosylation modification method comprises the following steps: (1) obtaining a recombinant protein Htt(1-90), to be specific, a. obtaining a hungtintin gene, b. constructing a recombinant expression plasmid pTWIN1-htt, c. constructing a recombinant escherichia coli strain DH5 alpha-htt for expressing the hungtintin, and d. expressing the recombinant protein; (2) purifying the recombinant protein Htt(1-90); (3) obtaining a glycosylation modified Htt(Cys-K92-K158) polypeptide by using an SPPS (stable protein plasma solution) method; (4) purifying the Htt(Cys-K92-K158) polypeptide; (5) coupling the Htt(Cys-K92-K158) polypeptide with the recombinant protein Htt(1-90) so as to obtain a glycosylation modified target protein Htt(1-158); (6) purifying the glycosylation modified target protein Htt(1-158). The glycosylation modification on the hungtintin shows that the glycosylated hungtintin has relatively strong stability; the influence of the temperature and the time on absorption peaks of the hungtintin shows that the hungtintin subjected to glycosylation modification 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)
1. a glycosylation modified method for Huntington protein, is characterized in that, 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.
2. the glycosylation modified method of Huntington protein according to claim 1, is characterized in that, 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.
3. the glycosylation modified method of Huntington protein according to claim 1, is characterized in that, 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.
4. the glycosylation modified method of Huntington protein according to claim 1, it is characterized in that, 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).
5. the glycosylation modified method of Huntington protein according to claim 3, is characterized in that, synthetic Cys-S138-K158 fragment solid phase carrier used is wang resin.
6. the glycosylation modified method of Huntington protein according to claim 1, is characterized in that, synthetic Cys-E110-L136 fragment and Cys-K92-I108 fragment solid phase carrier used are N-acyl group-benzoglyoxaline ketone resins.
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| 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》 * |
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