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 strepto...

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Bibliographic Details
Published in:Protein expression and purification 2005-03, Vol.40 (1), p.86-90
Main Authors: Jana, Snehasis, Karan, Goutam, Deb, J.K.
Format: Article
Language:English
Subjects:
Affinity chromatography
Chromatography, Affinity
Escherichia coli - genetics
Nucleotidyltransferases - genetics
Nucleotidyltransferases - isolation & purification
Nucleotidyltransferases - metabolism
Recombinant Fusion Proteins - isolation & purification
Recombinant Fusion Proteins - metabolism
Streptomycin adenylyltransferase
Thioredoxins - metabolism
Trx-His6-tagged fusion system
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Summary: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.
ISSN:1046-5928
1096-0279
DOI:10.1016/j.pep.2004.10.005