Purification of recombinant proteins from Escherichia coli and other model organisms is an essential component of biochemical and structural biology research. Over the past few decades, numerous strategies have been developed to allow easy purification of recombinant proteins by addition of a fusion protein or protein tag that allows rapid affinity purification. For some proteins, the fusion or tag poses a problem as the additional peptide sequences can interfere with function of the protein in biochemical assays or prevent the protein from forming compact crystals. If this occurs, the tag must often be removed after purification. This process of tag removal can be a cumbersome process.
Recently, a cysteine protease domain (CPD) embedded in a large protein toxin of Vibrio cholerae and other bacterial organisms was discovered that is inactive until induced by addition of the chemical compound inositol hexakisphosphate, also known as inositol-6-phosphate, InsP6, IP6, or phytic acid. Here, it is shown that a target protein can be purified as part of a fusion protein that includes the target protein fused to the CPD and a C-terminal peptide tag of 6-histidine residues. After purification of the fusion protein, the inducer molecule InsP6 is added and the protease and the peptide tag are removed from the fusion protein via autoproteolysis. The remaining portion of the fusion protein includes the target protein and an additional alanine and leucine residue added to the C-terminus of the target protein.
These disclosed strategies for purification of recombinant proteins followed by removal of a peptide tag may be adapted into any available cloning or purification systems. The molecule InsP6 is not produced by bacteria, hence, this strategy may be desirable for any recombinant protein produced in E. coli or other bacterial expression systems. This strategy also is desirable for any recombinant protein produced in plant, fungal, insect, or animal host cell expression systems in which the host cell is modified to block synthesis of InsP6.