Heterologous Escherichia coli expression, purification and characterization of the GrmA aminoglycoside-resistance methyltransferase.
The mechanism of resistance to aminoglycosides based on methylation of their target, 16S rRNA, was until recently described only in antibiotic producing microorganisms. However, equivalent methyltransferases have now also been identified among numerous clinical Gram-negative pathogenic isolates. We have cloned, expressed, and purified GrmA, the aminoglycoside-resistance methyltransferase from Micromonospora purpurea, producer of gentamicin complex. Two vectors were created that express protein with an N-terminal 6x histidine tag with and without an enterokinase recognition producing proteins His(6)-EK-GrmA and His(6)-GrmA, respectively. The activity of both recombinant proteins was demonstrated in vivo. After optimized expression and native purification both protein variants proved to be active in in vitro methylation assays. This work lays a foundation for future detailed biochemical, structural and pharmacological studies with this member of an important group of aminoglycoside-resistance enzymes.
Moric
Ivana
I
Institute of Molecular Genetics and Genetic Engineering, Laboratory for Molecular Genetics of Actinomycetes, University of Belgrade, Vojvode Stepe 444a, P.O. Box 23, 11010, Beograd, Serbia.
Bajkic
Sanja
S
Savic
Miloje
M
Tomic
Tatjana Ilic
TI
Conn
Graeme L
GL
Vasiljevic
Branka
B
D000617:Aminoglycosides / Q000494:pharmacology*; D001426:Bacterial Proteins; D003001:Cloning, Molecular / Q000379:methods*; D004351:Drug Resistance; D004926:Escherichia coli / Q000235:genetics*; D006639:Histidine; D008780:Methyltransferases / Q000096:biosynthesis* / Q000302:isolation & purification; D008847:Micromonospora / Q000201:enzymology
D000617:Aminoglycosides; D001426:Bacterial Proteins; D006639:Histidine; D008780:Methyltransferases