Journal
BIOTECHNOLOGY AND APPLIED BIOCHEMISTRY
Volume 37, Issue -, Pages 183-186Publisher
WILEY
DOI: 10.1042/BA20020044
Keywords
foldase; natural histidine tag; purification under denaturing conditions
Ask authors/readers for more resources
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 6 x His-tagged proteins from Escherichia coli. Given its ability to bind weakly to nickel-nitrilotriacetic acid (Ni2+-NTA), protocols for the purification of SlyD currently use an initial nondenaturing 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 Ni2+-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.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available