The effect of the hexahistidine-tag in the oligomerization of HSC70 constructs.
The hexahistidine is a fusion tag used for the isolation of proteins via an immobilized metal-ion affinity chromatography (IMAC). In the present study, we have purified and analyzed two constructs of the heat shock protein HSC70 in the presence or the absence of the His-tag (C30WT-His(+)/C30WT and C30DeltaL-His(+)/C30DeltaL). The oligomerization properties of the constructs were analyzed by size exclusion chromatography (SEC) and analytical ultracentrifugation (AU). Results from SEC analysis indicated that the His-tag promotes the dimerization of C30DeltaL-His(+) but has no effect on the elution profile of C30WT-His(+), compared to their respective untagged forms C30DeltaL and C30WT. These observations were also confirmed by AU analysis which indicates that C30DeltaL is stabilized in the dimeric form in the presence of the His-tag. These results emphasize the need to remove the His-tag before structural characterization of some recombinant proteins.
Amor-Mahjoub
Mouna
M
Laboratory of Biochemistry of Cellular and Molecular Signals, FRE 2621, CNRS-University P. & M. Curie, 96 Bd Raspail, 75006 Paris, France. mounamor@yahoo.fr <mounamor@yahoo.fr>
Suppini
Jean-Philippe
JP
Gomez-Vrielyunck
Nathalie
N
Ladjimi
Moncef
M
D000818:Animals; D002846:Chromatography, Affinity / Q000379:methods; D002850:Chromatography, Gel / Q000379:methods; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D018840:HSP70 Heat-Shock Proteins / Q000032:analysis / Q000737:chemistry* / Q000235:genetics; D006639:Histidine / Q000737:chemistry*; D008958:Models, Molecular; D050505:Mutant Proteins / Q000032:analysis / Q000737:chemistry / Q000235:genetics; D009154:Mutation / Q000235:genetics; D009842:Oligopeptides / Q000737:chemistry*; D051381:Rats; D011994:Recombinant Proteins / Q000032:analysis / Q000737:chemistry
D018840:HSP70 Heat-Shock Proteins; C471213:His-His-His-His-His-His; D050505:Mutant Proteins; D009842:Oligopeptides; D011994:Recombinant Proteins; D006639:Histidine