Single-step purification of a protein-folding catalyst, the SlyD peptidyl prolyl isomerase (PPI), from cytoplasmic extracts of Escherichia coli.
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.
Mukherjee
Sourav
S
Institute of Microbial Technology, Chandigarh-160 036, India.
Shukla
Anshuman
A
Guptasarma
Purnananda
P
D000595:Amino Acid Sequence; D002352:Carrier Proteins / Q000096:biosynthesis / Q000302:isolation & purification*; D002846:Chromatography, Affinity / Q000379:methods*; D003593:Cytoplasm / Q000737:chemistry* / Q000201:enzymology*; D004926:Escherichia coli / Q000737:chemistry* / Q000201:enzymology*; D029968:Escherichia coli Proteins; D008969:Molecular Sequence Data; D008970:Molecular Weight; D019696:Peptidylprolyl Isomerase / Q000096:biosynthesis / Q000302:isolation & purification*; D011487:Protein Conformation; D017510:Protein Folding; D020836:Protein Structure, Quaternary
D002352:Carrier Proteins; D029968:Escherichia coli Proteins; C085606:SlyD protein, E coli; D019696:Peptidylprolyl Isomerase