Strategies for folding of affinity tagged proteins using GroEL and osmolytes

• Published in: Journal of structural and functional genomics Vol. 10; no. 1; pp. 57 - 66
• Main Authors: Katayama, Hiroo, McGill, Mitchell, Kearns, Andrew, Brzozowski, Marek, Degner, Nicholas, Harnett, Bliss, Kornilayev, Boris, Matković-Čalogović, Dubravka, Holyoak, Todd, Calvet, James P, Gogol, Edward P, Seed, John, Fisher, Mark T
• Format: Journal Article
• Language: English
• Published: Dordrecht Dordrecht : Springer Netherlands 01-03-2009; Springer Netherlands; Springer Nature B.V
• Subjects: Binding Sites; Biochemistry; Bioinformatics; Biomedical and Life Sciences; Cells, Cultured; Chaperonin 60 - chemistry; Chaperonin 60 - genetics; Chaperonin 60 - metabolism; Chaperonins - chemistry; Chaperonins - metabolism; Dehydrogenases; E coli; Experiments; Forestry Management; Human Genetics; Humans; Life Sciences; Microbial Genetics and Genomics; Models, Molecular; Phosphoenolpyruvate Carboxykinase (ATP) - chemistry; Phosphoenolpyruvate Carboxykinase (ATP) - metabolism; Plant Genetics and Genomics; Plasmids; Protein Conformation; Protein Folding; Proteomics - methods; Recombinant Fusion Proteins - chemistry; Time Factors; Chaperonin; Protein stability; Affinity tags; Protein folding; Osmolytes
• ISSN: 1345-711X; 1570-0267
• DOI: 10.1007/s10969-008-9053-8

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.