title: GST-His purification: a two-step affinity purification protocol yielding full-length purified proteins.
abstract: Key assays in enzymology for the biochemical characterization of proteins in vitro necessitate high concentrations of the purified protein of interest. Protein purification protocols should combine efficiency, simplicity and cost effectiveness. Here, we describe the GST-His method as a new small-scale affinity purification system for recombinant proteins, based on a N-terminal Glutathione Sepharose Tag (GST) and a C-terminal 10xHis tag, which are both fused to the protein of interest. The latter construct is used to generate baculoviruses, for infection of Sf9 infected cells for protein expression. GST is a rather long tag (29 kDa) which serves to ensure purification efficiency. However, it might influence physiological properties of the protein. Hence, it is subsequently cleaved off the protein using the PreScission enzyme. In order to ensure maximum purity and to remove the cleaved GST, we added a second affinity purification step based on the comparatively small His-Tag. Importantly, our technique is based on two different tags flanking the two ends of the protein, which is an efficient tool to remove degraded proteins and, therefore, enriches full-length proteins. The method presented here does not require an expensive instrumental setup, such as FPLC. Additionally, we incorporated MgCl2 and ATP washes to remove heat shock protein impurities and nuclease treatment to abolish contaminating nucleic acids. In summary, the combination of two different tags flanking the N- and the C-terminal and the capability to cleave off one of the tags, guaranties the recovery of a highly purified and full-length protein of interest.
authors.affiliation: Genome Stability Laboratory, Oncology Axis, Hôtel-Dieu de Québec.
authors.forename: Ranjan
authors.initials: R
authors.lastname: Maity
authors.forename: Joris
authors.initials: J
authors.lastname: Pauty
authors.forename: Jana
authors.initials: J
authors.lastname: Krietsch
authors.forename: Rémi
authors.initials: R
authors.lastname: Buisson
authors.forename: Marie-Michelle
authors.initials: MM
authors.lastname: Genois
authors.forename: Jean-Yves
authors.initials: JY
authors.lastname: Masson
mesh_terms: D002846:Chromatography, Affinity / Q000379:methods*; D005978:Glutathione / Q000737:chemistry*; D006639:Histidine / Q000737:chemistry*; D011994:Recombinant Proteins / Q000302:isolation & purification*
chemical_list: D011994:Recombinant Proteins; D006639:Histidine; D005978:Glutathione