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Single-step purification of a protein-folding catalyst, the SlyD peptidyl prolyl isomerase (PPI), from cytoplasmic extracts of Escherichia coli.

DIR@IMTECH: CSIR-Institute of Microbial Technology

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Title Single-step purification of a protein-folding catalyst, the SlyD peptidyl prolyl isomerase (PPI), from cytoplasmic extracts of Escherichia coli.
 
Creator Mukherjee, Sourav
Shukla, Anshuman
Guptasarma, Purnananda
 
Subject QR Microbiology
 
Description The protein-folding catalyst SlyD, a peptidyl prolyl cis-trans isomerase regulated by metal binding, was initially discovered as a major contaminant of non-denaturing immobilized metal-affinity chromatography (IMAC)-based procedures for the purification of heterologously expressed 6xHis-tagged proteins from Escherichia coli. Given its ability to bind weakly to nickel-nitrilotriacetic acid (Ni(2+)-NTA), protocols for the purification of SlyD currently use an initial non-denaturing IMAC step, followed by an ion-exchange chromatographic step and occasionally also other chromatographic steps. Here we demonstrate that using denaturing conditions instead of non-denaturing conditions, and by processing of large quantities of culture through small volumes of Ni(2+)-NTA resin to increase competition for binding, single-step purification of SlyD to homogeneity can be achieved directly from E. coli extracts, as assessed through spectroscopic and electrophoretic methods. The purified, denatured SlyD is shown to be capable of refolding to give rise to a native-like CD spectrum, establishing the utility of the procedure. The procedure also establishes SlyD to be the only E. coli protein capable of contaminating denaturing IMAC-based procedures.
 
Publisher Portland Press
 
Date 2003-04
 
Type Article
PeerReviewed
 
Relation http://www.babonline.org/bab/037/0183/bab0370183.htm
http://crdd.osdd.net/open/250/
 
Identifier Mukherjee, Sourav and Shukla, Anshuman and Guptasarma, Purnananda (2003) Single-step purification of a protein-folding catalyst, the SlyD peptidyl prolyl isomerase (PPI), from cytoplasmic extracts of Escherichia coli. Biotechnology and applied biochemistry, 37 (Pt 2). pp. 183-6. ISSN 0885-4513