Strategies for folding of affinity tagged proteins using GroEL and osmolytes
- 159 Downloads
Obtaining a proper fold of affinity tagged chimera proteins can be difficult. Frequently, the protein of interest aggregates after the chimeric affinity tag is cleaved off, even when the entire chimeric construct is initially soluble. If the attached protein is incorrectly folded, chaperone proteins such as GroEL bind to the misfolded construct and complicate both folding and affinity purification. Since chaperonin/osmolyte mixtures facilitate correct folding from the chaperonin, we explored the possibility that we could use this intrinsic binding reaction to advantage to refold two difficult-to-fold chimeric constructs. In one instance, we were able to recover activity from a properly folded construct after the construct was released from the chaperonin in the presence of osmolytes. As an added advantage, we have also found that this method involving chaperonins can enable researchers to decide (1) if further stabilization of the folded product is required and (2) if the protein construct in question will ever be competent to fold with osmolytes.
KeywordsChaperonin Osmolytes Affinity tags Protein folding Protein stability
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
Small ubiquitin-like modifier
This research was supported in part by NSF MCB-0445936 (MTF), NIH R41 GM080074 (MTF), NIH P50 DK057301 (JPC), and a grant from the Kansas Technology Enterprise Corporation KTEC (MTF). MB and AK are undergraduates supported by the NSF grant. AK was specifically supported by an NSF REU supplement NSF MCB-0735909 (MTF). A portion of this research was presented at the “Protein Structure Inititative “Bottlenecks” Workshop. The authors gratefully acknowledge Stephen C. Parnell for generating the pSUMO-HT193 plasmid and for helpful discussions with D. M.-C. D. M.-C. was supported by a Fulbright Scholarship.