Purification of streptomycin adenylyltransferase from a recombinant Escherichia coli.
Bacterial resistance to aminoglycosides continues to escalate and is widely recognized as a serious health threat, contributing to interest in understanding the mechanisms of resistance. One important mechanism of streptomycin modification is through ATP dependent O-adenylation, catalyzed by streptomycin adenylyltransferase (SMATase). The aim of this study was to purify the recombinant SMATase by Ni(2+)-IDA-His bind resin column chromatography. Thioredoxin-His6-tagged SMATase fusion protein was produced in a bacterial intracellular expression system mainly in a soluble form. The purified fusion protein showed a single band on SDS-PAGE corresponding to 49 kDa. The recovery of fusion protein was 47% with ninefold purification. The fusion system provided a single step, easy and very rapid purification of SMATase and is suitable for obtaining a highly purified functional protein of interest. The fusion does not affect the functionality of the protein.
Jana
Snehasis
S
Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology-Delhi, Hauz Khas, New Delhi 110016, India.
Karan
Goutam
G
Deb
J K
JK
D002846:Chromatography, Affinity; D004926:Escherichia coli / Q000235:genetics*; D009713:Nucleotidyltransferases / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D013879:Thioredoxins / Q000378:metabolism
D011993:Recombinant Fusion Proteins; D013879:Thioredoxins; D009713:Nucleotidyltransferases; C022678:streptomycin 3''-adenylyltransferase