Recovery of Polypeptides Cleaved from Purified Calmodulin-Binding Peptide Fusion Proteins

Purification of recombinant proteins has been greatly simplified in recent years because of the availability of expression vectors that allow fusion of the protein coding sequence of interest to short peptide sequences or larger proteins, enabling the affinity purification of the fusion protein from crude preparations. Fusion proteins are often engineered to contain short linkers of five to six amino acids, positioned between the affinity tag and the protein of interest, which serve as recognition targets for site-specific proteases thereby allowing the proteolytic removal of the affinity tag following purification of the fusion protein. The serine protease enterokinase (EK) is particularly attractive because it cleaves after the carboxy terminus of its recognition sequence (Asp)4Lys, allowing production of cleavage products that have native amino termini following the removal of N-terminal affinity tags. In addition, the recent cloning of bovine EK and its expression and purification from E. coli has allowed the production of high specific activity enzyme that is virtually free of contaminating proteases (1,5). At a low enzyme-to-substrate ratio (1:1000 wt/ wt), fusion protein can often be cleaved to completion in a few minutes at 37°C, whereas with other site-specific proteases such as thrombin and factor Xa, cleavage reactions are usually carried out over a period of several hours.