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Phosphorylation modification method of huntingtin protein

A technology of huntingtin protein and modification method, applied in the field of chemical modification of proteins, can solve problems such as easy aggregation

Inactive Publication Date: 2016-08-17
杭州拜善晟生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] 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

Method used

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  • Phosphorylation modification method of huntingtin protein
  • Phosphorylation modification method of huntingtin protein
  • Phosphorylation modification 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 lig...

Embodiment 2

[0040] Embodiment 2: Purification of recombinant protein Htt(1-90)

[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.

[0042] 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 40°C for 16h. Solution C (Na-HEPES (pH8.0) 20mmol / L, NaCl 500mmol / 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%.

[0043] A solution c...

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Abstract

The invention discloses a phosphorylation modification method of hungtintin. The phosphorylation modification method of the hungtintin 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 phosphorylation 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 phosphorylation modified target protein Htt(1-158); (6) purifying the phosphorylation modified target protein Htt(1-158). The phosphorylation modified hungtintin has the advantages that the aggregation of the hungtintin is reduced, the aggregation time of the hungtintin is also improved, after aggregation, the length and the height of the hungtintin are improved, the aggregation of the hungtintin is obviously delayed, and the toxic and side effects of the hungtintin on nerve cells are hindered very well.

Description

technical field [0001] The invention relates to a chemical modification of protein, in particular to a phosphorylation 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. As an important way of post-translational modification of proteins, protein phosphorylation is the most basic, common and important mechanism to regulate and control protein activity and function. Protein phosphorylation mainly occurs on two amino acids, one is serine (Thr) and tryptophan (Ser), and the other is tyrosine. The phosphorylation of protein affects the folding and aggregation of protein, thereby affecting its tertiary structure, and then affecting the function of protein, especially for some kinases (mostly...

Claims

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Application Information

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IPC IPC(8): C07K14/47C07K1/107C12N15/70
CPCC07K14/47C12N15/70
Inventor 王喆明
Owner 杭州拜善晟生物科技有限公司
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