Methyltransferase ErmC`: Gene Cloning and Expression and Protein Purification (CROSBI ID 474161)
Prilog sa skupa u zborniku | sažetak izlaganja sa skupa | domaća recenzija
Podaci o odgovornosti
Maravić, Gordana ; Flögel, Mirna
engleski
Methyltransferase ErmC`: Gene Cloning and Expression and Protein Purification
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.
Methyltransferase ErmC`; Antibiotics; Resistance
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Podaci o prilogu
77-77-x.
2000.
objavljeno
Podaci o matičnoj publikaciji
Book of Abstracts
Flögel, M. i sur
Zagreb: Hrvatsko biokemijsko društvo
Podaci o skupu
HB2000, Silver Jubilee Meeting of the Croatian Biochemical Society
poster
13.10.2000-15.10.2000
Zagreb, Hrvatska