Cloning and Prokaryotic Expression of C-type Lysozyme Gene from Agrius convolvuli
Autor(es): Kim, Jong-Wan; Yoe, Sung Moon
Resumo: 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 Xhol was ligated into the pGEX 4T-1 vector, which contained the glutathione S-transferase (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.
Palavras-Chave: :recombinant lysozyme; bacterial expression; glutathione S-transferase; Agrius convolvuli
Imprenta: Animal Cells and Systems, v. 12, n. 3, p. 149-155, 2008
Identificador do objeto digital: 10.1080/19768354.2008.9647168
Descritores: Aedes aegypti - Molecular screening
Data de publicação: 2008
