Strategies for folding of affinity tagged proteins using GroEL and osmolytes.
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.
Katayama
Hiroo
H
Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, KS, USA.
McGill
Mitchell
M
Kearns
Andrew
A
Brzozowski
Marek
M
Degner
Nicholas
N
Harnett
Bliss
B
Kornilayev
Boris
B
Matković-Calogović
Dubravka
D
Holyoak
Todd
T
Calvet
James P
JP
Gogol
Edward P
EP
Seed
John
J
Fisher
Mark T
MT
D001665:Binding Sites; D002478:Cells, Cultured; D018834:Chaperonin 60 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018833:Chaperonins / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D019764:Phosphoenolpyruvate Carboxykinase (ATP) / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D017510:Protein Folding; D040901:Proteomics / Q000379:methods; D011993:Recombinant Fusion Proteins / Q000737:chemistry*; D013997:Time Factors
D018834:Chaperonin 60; D011993:Recombinant Fusion Proteins; D018833:Chaperonins; D019764:Phosphoenolpyruvate Carboxykinase (ATP)