Recombinant Protein Purification Techniques

The gene encoding protein Stl was expressed from a pGEX-4T-1 vector (GE Healthcare, Chicago, Illinois, USA) as a glutathione S-transferase (GST)-fused construct. The GST-tag was cleaved from the protein via overnight thrombin digestion during its purification process [24 (link)]. The E. coli dUTPase gene was inserted into a pET-15b vector (Merck KGaA, Darmstadt, Germany) between the BamHI and NdeI cleavage sites to enable its expression with an N-terminal His-tag which was used in further purification steps [32 (link)]. Both protein Stl and our E. coli dUTPase constructs were expressed in E. coli BL21 (DE3) Rosetta cells (Novagen) and they were purified according to our previously used protocol [24 (link)].