Naslov
Methyltransferase ErmC`: Gene Cloning and Expression and Protein Purification

Autori
Maravić, Gordana ; Flögel, Mirna

Vrsta, podvrsta i kategorija rada
Sažeci sa skupova, sažetak, znanstveni

Izvornik
Book of Abstracts / Flögel, M. i sur - Zagreb : Hrvatsko biokemijsko društvo, 2000, 77-77

Skup
HB2000, Silver Jubilee Meeting of the Croatian Biochemical Society

Mjesto i datum
Zagreb, Hrvatska, 13.10.2000. - 15.10.2000

Vrsta sudjelovanja
Poster

Vrsta recenzije
Domaća recenzija

Ključne riječi
Methyltransferase ErmC`; Antibiotics; Resistance

Sažetak
Bacterial resistance to macrolide-lincosamide-streptogramin B 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.

Izvorni jezik
Engleski

Znanstvena područja
Biologija

POVEZANOST RADA