Glutathione sepharose 4b affinity chromatography

GST, GST-ANKR, and GST-GGA3 proteins were expressed in Escherichia coli (BL21(DE3)). Cells grown in LB medium were incubated in the presence of 0.1 mM isopropyl-1-thio-β-D-galactopyranoside (15 hours at 25°C). After centrifugation, resulting cell pellets were resuspended in a buffer containing a mixture of protease inhibitors (20 mM Tris-HCl pH 7.5, 1 mM EDTA, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 50 units/mL aprotinin, 2 μg/mL leupeptin, and 2 μg/mL pepstatin A) and subjected to sonication. The cell debris was removed by centrifugation and the resultant supernatant was used as an E. coli lysate. Purification of GST fusion proteins to near homogeneity was achieved using Glutathione-Sepharose 4B affinity chromatography (GE Healthcare, Piscataway, NJ). The purity of the samples was at least 80%, as judged by Coomassie Brilliant Blue staining of SDS-polyacrylamide gel electrophoresis (PAGE).

The Ov-16 nucleotide sequence [18 (link)], without the signal peptide, was amplified by PCR from a recombinant pMAL-Ov-16 plasmid, kindly donated by Professor JE Bradley, School of Life Sciences and University of Nottingham, UK. A poly-His tail was added to the carboxy-terminus of the amplified Ov-16 sequence, which was then subcloned into pGEX-6P-1 plasmid (GE Healthcare, Little Chalfont, UK), a glutathione S-transferase fusion vector. The GST-Ov-16 recombinant protein was purified by Glutathione Sepharose 4B affinity chromatography (GE Healthcare) according to the manufacturer’s recommendations. Later, separation of the recombinant protein from the glutathione S-transferase moiety was accomplished by site-specific proteolysis using PreScission^TM Protease reaction (GE Healthcare). After dialyzing against PBS, the Ov-16-His recombinant protein was repurified by Ni²⁺ Sepharose 4B affinity chromatography (GE Healthcare) according to the manufacturer’s recommendations. Finally, the Ov-16-His was again dialyzed against PBS and quantified with Pierce™ BCA Protein Assay Kit (Thermo Scientific, Rockford, IL, USA).