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Acylation modification method of hungtintin

A technology of huntingtin protein and modification method, applied in the field of chemical modification of proteins, can solve the problems of easy aggregation and low degree of proteolysis, etc.

Inactive Publication Date: 2014-02-26
杭州璞题生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Unmodified huntingtin protein shows that it is easier to aggregate, etc. At present, there are no research institutes or institutions in China to chemically modify huntingtin protein. Through acylation chemical modification of huntingtin protein, the degree of proteolysis after acetylation treatment is small, rather than The greater degree of hydrolysis of acetylated protein indicates that the stability of acetylated huntingtin protein is stronger

Method used

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  • Acylation modification method of hungtintin
  • Acylation modification method of hungtintin
  • Acylation modification method of hungtintin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1 : obtain recombinant protein Htt1-90

[0027] (1) Test materials

[0028] (1) Vectors and strains

[0029] The plasmid pcDNA3.1 / mys-His was donated by CHDI; the expression vector pTWIN1 was purchased from NEB Company; the plasmid pMD18-T and Escherichia coli JM109 and DH5α were purchased from Takara Biotechnology Co., Ltd.

[0030] (2) Primers

[0031] P1: 5-CCG GAATTC CTGCCGTGCC-3 ( Eco R?)

[0032] P2: 5-AAAA CTGCAG ACAGCCGGGC-3 ( Pst ?)

[0033] Synthesized by Shanghai Sangon Bioengineering Co., Ltd.

[0034] (2) Implementation plan

[0035] 1. Obtaining the Huntingtin Gene

[0036] Using the plasmid pcDNA3.1 / mys-His as a template, design and synthesize Eco R? and Pst ?Upstream and downstream primers P1 and P2 of restriction enzyme sites, PCR amplification htt Gene fragments, PCR products were detected by agarose gel electrophoresis and recovered, purified and ligated with the vector pMD18-T, transformed into Escherichia coli JM109, an...

Embodiment 2

[0042] Example 2 : Purification of recombinant protein

[0043] The CBD component of the Htt1-90-Intein-CBD fusion protein can be specifically combined with chitin resin to facilitate the removal of foreign proteins, and then the intein component catalyzes the fusion protein to break between Htt1-90 and Intein under certain conditions. The specific operation is as follows: the supernatant is loaded on a 2ml chitin gravity column. The column was first equilibrated with solution A (Na-HEPES (pH8.0) 20mmol / L, NaCl 500mmol / L, EDTA 1mmol / L), and the supernatant was loaded and then eluted with solution A. Then the column was soaked in solution B (Na-HEPES (pH8.0) 20mmol / L, NaCl 500mmol / L, EDTA 1mmol / L, DTT 50mmol / L) at 40C for 16h. Solution C (Na-HEPES (pH8.0) 20mmol / L, NaCl 500mmol / L) eluted protein and collected every 1ml. The samples collected at each step were analyzed by SDS-PAGE, and the purity of the identified protein was 90%.

[0044] A solution containing 0.25mmol / L p...

Embodiment 3

[0045] Example 3 : SPPS method to obtain Htt (Cys-K92-K158) polypeptide modified by acylation at the K92 site

[0046] The synthesis process is divided into three fragments, first synthesizing Cys-S138-K158, then synthesizing Cys-E110-L136, and finally synthesizing Cys-K92-I108. Each fragment is ligated by natural chemical ligation NCL method to obtain Htt (Cys-K92-K158) polypeptide

[0047] The synthesis of solid-phase peptides was performed on a CS336X peptide synthesizer, and the synthesis of Cys-S138-K158 peptides was based on Wang resin. The deprotection and depolymerization of the resin can proceed spontaneously in the solution of (TFA / DCM / H2O / TIPS 90 / 5 / 2.5 / 2.5). Then it was precipitated by 10 times equivalent of cold ether, and these original polypeptides were purified by reverse phase HPLC, using waters 600 pump and waters 2489 UV / visible light detector, and the column used GRACE-Vydac 218TP54 C18 column. Mobile phase A is 0.1% trifluoroacetic acid aqueous solution...

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Abstract

The invention discloses an acylation modification method of hungtintin. The acylation modification method is characterized by comprising 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 an acylation 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 an acylation modified target protein Htt(1-158); (6) purifying the acylation modified target protein Htt(1-158). Through the acylation chemical modification on the hungtintin, the hydrolysis degree of the hungtintin subjected to the acylation treatment is relatively small, and the hydrolysis degree of the non-acetylized hungtintin is relatively high, which shows that the hungtintin subjected to the acylation modification has relatively strong stability.

Description

technical field [0001] The invention relates to a chemical modification of protein, in particular to an acylation modification method of huntingtin protein. Background technique [0002] Protein modification is a complex process. There are many types of modification in eukaryotes. The common ones are glycosylation, acetylation, ubiquitination, phosphorylation and SUMOylation. Protein modification can change the activity, location or function of proteins. Acetylation modification in proteins can regulate various properties of proteins, including DNA-protein interactions, subcellular localization, transcriptional activity, and protein stability. Acetylated proteins are highly resistant to degradation by aminopeptidases and ubiquitin-dependent proteases. Studies have shown that the imbalance of protein acetylation and deacetylation is a key process in the expansion of polyglutamine-induced diseases. [0003] Huntington's disease (HD) is an autosomal dominant neurodegenerative...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/47C07K1/107C12N15/70
CPCC07K14/47C12N15/70
Inventor 王喆明
Owner 杭州璞题生物科技有限公司
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