Construction of histone H3K4M peptide fused EML3 plasmid and fusion protein purification
This paper aims to develop a method for obtaining EML3 fusion protein containing N-terminal or C-terminal fusion with histone H3K4M peptide.The encoding region of H3 peptide(aa 1-15)was fused into the recombinant plasmid expressing EML3 Agenet domain,resulting in the fusion plasmid expressing H3K4M fusion protein at N-or C-terminals.The plasmid was then transformed into Escherichia coli BL21(DE3).After expansion culture,the expression of the 6×His-tagged EML3 fusion protein was induced at 15℃by adding IPTG.The purity of the protein was assessed by SDS-PAGE and coomassie brilliant blue staining,after purification by nickel affinity chromatography and gel filtration chromatography.Through plasmid site-directed mutagenesis,plasmid restriction digestion,PCR amplification,and homologous recombination,we successfully constructed the recombinant plasmid of EML3 fusion protein containing N-or C-terminal fusion with H3K4M peptide.Sequencing confirmed the correct sequence,and the recombinant plasmid successfully induced the expression of the fusion protein in Escherichia coli.The C-terminal fusion protein(EML3-H3K4M)was cleaved by thrombin,resulting in the release of free H3K4M peptide as well as the EML3 Agenet domain protein.We successfully constructed the recombinant plasmid of EML3 fused with histone H3K4M peptide and obtained a highly pure EML3 fusion protein by IPTG induction and protein purification.
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