engleski

Methyltransferase ErmC' gene cloning and expression and protein purification

Bacterial resistance to macrolide-lincosamide-streptogramin B (MLS) antibiotics is based on activity of methyltransferases of Erm family. Erm methyltransferases mono- and dimethylate specific adenine residue within 23S rRNA, thus preventing antibiotic binding to the ribosome. Here we describe the optimum procedure for the expression, isolation and purification of ErmC', which is biochemically more stabile than most members of Erm family. ermC' gene was isolated and amplified from plasmid pIM13 from Bacillus subtilis BD1167 via polymerase chain reaction. Simultaneously, new restriction sites were introduced at both ends of the gene to enable its cloning to an expression vector pET-25b(+). Expression host strain BL21(DE3)pLysS was then transformed with recombinant vector pET-25b(+)ermC'. Protein expression was induced with IPTG and optimized for obtaining soluble protein at a greater rate. Optimum procedure for purification of protein from bacterial protein extract was designed after trying of several purification methods. The procedure involves two subsequent steps of cation exchange, with use of stepwise salt gradient in the first step, and a linear salt gradient in a second one. Highly purified ErmC' has been obtained in large quantities to be used in future studies of ErmC' activity, as a contribution to the intensive worldwide efforts to find potent and specific inhibitors for overcoming the MLS resistance.

MLS antibiotics; 23S rRNA; Erm methyltransferases; ErmC; ErmC’; ErmAM; MLS resistance; inhibitors of Erm methyltransferases

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