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Preparation method of human protein lysine methyltransferase SET9 fusion protein

A technology of lysine methyl and fusion protein is applied in the field of genetic engineering Escherichia coli fermentation to prepare recombinant human SET9 fusion protein, which can solve the problems of insolubility and difficulty in increasing active SET9 protein extraction.

Inactive Publication Date: 2012-10-10
SOUTHEAST UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The fusion protein expression of histidine (His) tag has the problem of insolubility, and it needs to be denatured during purification. Therefore, the active protein can only be obtained through the renaturation process, which increases the difficulty of extracting active SET9 protein

Method used

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  • Preparation method of human protein lysine methyltransferase SET9 fusion protein
  • Preparation method of human protein lysine methyltransferase SET9 fusion protein
  • Preparation method of human protein lysine methyltransferase SET9 fusion protein

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Experimental program
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Embodiment 1

[0014] 1. PCR amplification of SET9 gene fragment

[0015] Using pCMV-SET9 (purchased from Clontech, USA) as a template, the SET9 gene fragment containing the SET domain was amplified by PCR. The cDNA sequence of SET9 was determined by NCBI GenBank (accession No, AF448510), EcoRI and Xho I were selected as restriction sites, and DNA Star software was used to design SET9 (EcoRI---Xho I) PCR primers and upstream primers (SET9-f) The sequence is: 5'-ACCGAATTCCATGGATAGCGACGAC-3', containing the EcoR I restriction site (G↓AATTC) and the initiation codon (ATG); the sequence of the downstream primer (SET9-r) is: 5'-CTCCTCGAGTCATTACTTTTGCTGGGTGGCCTG-3', Contains a double stop codon (TCATTA) and an XhoI restriction site (C↓TCGAG), located at the 268th to 1365th nucleotide of the complete gene sequence of SET9. Product: Length=1098+10+15=1123bp, GC%=50.0.

[0016] reaction system:

[0017] Reagent

Volume(μL)

10x PCR Buffer (Pfu)

5

Pfu (2.5u / μL, Strata...

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Abstract

The invention discloses a preparation method of human protein lysine methyltransferase SET9 fusion protein. In the method, an SET9 gene segment is connected to the vector plasmid containing a variable-code glutathione S-transferase code reading frame, the host strain is transformed and subjected to inducible expression and then purified through a Glutathione Agarose adsorption column. Through the advantages that the T7RNA polymerase gene of escherichia coli BL21 (DE3) expression bacteria can be subjected to the inducible expression of IPTG (isopropyl-beta-d-thiogalactoside) and the decomposition of the expressed fusion protein can be inhibited since the bacteria do not have the membrane-bound protease activity and the like, according to the invention, a prokaryotic expression system of the GST-SET9 fusion protein is successfully established by transferring the recombinant vector pGEX-SET9 into the BL21 (DE3) expression bacteria, and the high-activity fusion protein is purified. The method disclosed by the invention can be applied to the laboratory investigation such as nonhistone protein methylation effect and SET9 antibody development.

Description

technical field [0001] The invention relates to the field of biotechnology. Specifically, DNA recombination technology is used to construct a high-efficiency prokaryotic expression system for human protein lysine methyltransferases (protein lysine methyltransferases, PLMTs or PKMTs) SET9 fusion protein, and a genetically engineered Escherichia coli fermentation A method for preparing recombinant human SET9 fusion protein. Background technique [0002] SET9, also known as histone lysine N-methyltransferase and histone H3-K4 methyltransferase, is a protein modification enzyme that converts S-adenosyl-L-methionine (S-adenosyl- The methyl group provided by L-methionine (SAM) is transferred to the lysine residue of the target protein to make it methylated, causing changes in protein structure and function and producing related biological effects. The full length of SET9 is 366 amino acids, the C-terminal contains a conserved SET domain, the molecular weight is about 50KD, and it...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/54C12N15/70C12N9/10
Inventor 余卫平
Owner SOUTHEAST UNIV
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