A simplified method for producing laboratory grade recombinant TEV protease from E. coli.

Abstract The tobacco etch virus (TEV) protease has become a popular choice for cleaving fusion proteins because of its high stringency in sequence recognition. Procedures for isolating recombinant protein from the cytoplasm of E. coli require rupturing of the cell wall via enzymatic treatment combined with sonication or French press. Here we present an expedited method for producing laboratory-grade TEV protease in E. coli using a freeze-thaw method, followed by purification with immobilized metal affinity chromatography. Protease is obtained by expression from the pDZ2087 plasmid in BL21 (DE3) cells. Proteolysis resulting from this product, cleaves a maltose-binding protein fusion to completion at a fusion-to-protease molar ratio of 50:1.