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Palmitoylation method of huntingtin protein

A huntingtin protein modification method technology, applied in the field of chemical modification of proteins, can solve the problems of protein aggregation, cell death and exacerbation of cytotoxicity

Inactive Publication Date: 2016-09-21
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

In contrast, mutation of the Htt palmitoylation site in PolyQ without palmitoylation exacerbated its cytotoxicity, resulting in protein aggregation and cell death

Method used

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  • Palmitoylation method of huntingtin protein
  • Palmitoylation method of huntingtin protein
  • Palmitoylation method of huntingtin protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1: Obtaining recombinant protein Htt1-90

[0025] 1) Test material

[0026] (1) Vector and strain

[0027] Plasmid pcDNA3.1 / myc-His was donated by CHDI; expression vector pTWIN1 was purchased from NEB Company; plasmid pMD18-T and Escherichia coli JM109, DH5α were purchased from Takara Biotechnology Co., Ltd.

[0028] (2) Primers

[0029] P1: 5-CCG GAATTC CTGCCGTGCC-3 (EcoR I)

[0030] P2: 5-AAAA CTGCAG ACAGCCGGGC-3 (Pst I)

[0031] Synthesized by Shanghai Sangon Bioengineering Co., Ltd.

Embodiment approach

[0033] 1. Obtaining the Huntingtin Gene

[0034] Using the plasmid pcDNA3.1 / myc-His as a template, design and synthesize upstream and downstream primers P1 and P2 containing EcoR I and Pst I restriction enzyme sites to amplify the htt gene fragment by PCR, and detect the PCR product by agarose gel electrophoresis Recovered, purified and ligated with vector pMD18-T, transformed into Escherichia coli JM109, and screened for ampicillin resistance to obtain recombinant plasmid pMD18T-htt, which was sent to Shanghai Sangon Bioengineering Co., Ltd. for sequencing. The verified recombinant plasmid was digested with EcoR I and Pst I, and the digested product was purified and stored in a refrigerator at 4°C.

[0035] 2. Construction of recombinant expression plasmid pTWIN1-htt

[0036] The prokaryotic expression vector pTWIN1 was double-digested with restriction endonucleases EcoR I and Pst I, and after purification, the linear pTWIN1 plasmid fragment and the htt gene fragment were li...

Embodiment 2

[0040] Embodiment 2: Purification of recombinant protein

[0041] 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 immersed 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, NaCl500mmol / L) eluted protein and collected every 1ml. The samples collected in each step were analyzed by SDS-PAGE, and the purity of the identified protein was 90%.

[0042] A solution containing 0.25 mmol / L...

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Abstract

The invention discloses a palmitoylation modification method of hungtintin. The palmitoylation 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 palmitoylation 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 palmitoylation modified target protein Htt(1-158); (6) purifying the palmitoylation modified target protein Htt(1-158). The palmitoylation modification on the hungtintin shows that the stability of the hungtintin is improved, the palmitoylation modified hungtintin has the capacity of resisting hydrolytic enzymes and relatively strong stability, and the research on the palmitoylation of the hungtintin has important significance for the treatment of Huntington diseases.

Description

technical field [0001] The present invention relates to a kind of chemical modification of protein, in particular to a kind of palmito-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 palmitoylation, acetylation, ubiquitination, phosphorylation and sumoylation. Protein modification can change the activity, location or function of proteins. Palmitization of proteins refers to the covalent binding of 16-carbon fatty acid palmitate to specific cysteine ​​residue side chains of proteins through thioester bonds, which is very important for the functional regulation of different membrane proteins and membrane protein signaling pathways. important. [0003] Huntington's disease (Huntington disease, HD) is an autosomal dominant neurodegenerative disease. The HD gene encodes a huntingtin protein (Huntingtin, Htt) consisting of 3144 amino acids. T...

Claims

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Application Information

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Patent Type & Authority Patents(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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