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Cloning and prokaryotic expression of C-type lysozyme gene from agrius convolvuli

Animal Cells and Systems 2008년 12권 3호 p.149 ~ 155
김종완, 여성문,
소속 상세정보
김종완 ( Kim Jong-Wan ) - Dankook University Department of Biological Sciences
여성문 ( Yoe Sung-Moon ) - Dankook University Department of Biological Sciences

Abstract


We have isolated and characterized Agrius convolvuli cDNA encoding a c?type lysozyme. The cDNA sequence encodes a processed protein of 139 amino acid residues with 19 amino acid residues amino?terminal signal sequence and 120 amino acid residues mature sequence. The amino acid residues responsible for the catalytic activity and the binding of the substrate are conserved. Agrius lysozyme has a high identity to Manduca sexta. Recombinant A. convolvuli lysozyme was expressed in Escherichia coli BL21 (DE3) pLysS cells for pGEX 4T?1 expression vector. Their optimal conditions for the fusion protein expression and purification were screened. Lysozyme gene amplified with primers ACLyz BamHI and ACLyz XhoI was ligated into the pGEX 4T?1 vector, which contained the glutathione Stransferase (GST) gene for fusion partner. The fusion protein was induced by IPTG and identified by SDS?PAGE analysis. Molecular weight of the fusion protein was estimated to be about 45 kDa. Recombinant lysozyme, fused to GST, was purified by glutathion?Sepharose 4B affinity chromatography. Western blot analysis of this protein revealed an immunoreactivity with the anti?Agrius lysozyme.

키워드

recombinant lysozyme; bacterial expression; glutathione S?transferase; Agrius convolvuli

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