The human GEFH1 (Uniprot Q92974–1) C1 (residues 28–100) and PH domains (residues 439–589) were initially cloned in isolation into a pET-based bacterial expression vector containing an N-terminal thioredoxin-6xHis cleavable tag using a restriction free positive selection method (Olieric et al., 2010 (link)). The GEFH1-C1-PH-GCN4 construct was assembled by homologous recombination using overlapping PCR fragments by fusing in frame the leucine zipper coiled-coil domain of the yeast transcriptional activator GCN4 (O’Shea et al., 1991 (link)) C-terminally to the PH domain. All clones were verified by sequencing.
Protein samples were produced by overexpression in E. coli Bl21(DE3) cells. Protein purification was performed by immobilized metal-affinity chromatography (IMAC) on HisTrap HP Ni2+ Sepharose columns (GE Healthcare) according to the manufacturer’s instructions. Processed protein samples were concentrated and processed on a HiLoad Superdex 200 16/60 size exclusion chromatography column (GE Healthcare) equilibrated in 50 mM Tris HCl, pH 7.5, supplemented with 150 mM NaCl and 2 mM DTT. Protein fractions were analyzed by Coomasie stained SDS-PAGE. Fractions containing the target protein were pooled and concentrated by ultrafiltration. Protein concentrations were estimated by UV absorbance at 280 nm.
Protein samples were produced by overexpression in E. coli Bl21(DE3) cells. Protein purification was performed by immobilized metal-affinity chromatography (IMAC) on HisTrap HP Ni2+ Sepharose columns (GE Healthcare) according to the manufacturer’s instructions. Processed protein samples were concentrated and processed on a HiLoad Superdex 200 16/60 size exclusion chromatography column (GE Healthcare) equilibrated in 50 mM Tris HCl, pH 7.5, supplemented with 150 mM NaCl and 2 mM DTT. Protein fractions were analyzed by Coomasie stained SDS-PAGE. Fractions containing the target protein were pooled and concentrated by ultrafiltration. Protein concentrations were estimated by UV absorbance at 280 nm.