Hui Chin Goh, Radoslaw M. Sobota, Farid J. Ghadessy, Saurabh Nirantar, Going native: Complete removal of protein purification affinity tags by simple modification of existing tags and proteases, Protein Expression and Purification, Volume 129, 2017, Pages 18-24, ISSN 1046-5928, https://doi.org/10.1016/j.pep.2016.09.001.
Protein purification typically involves expressing a recombinant gene comprising a target protein fused to a suitable affinity tag. After purification, it is often desirable to remove the affinity tag to prevent interference with downstream functions of the target protein. This is mainly accomplished by placing a protease site between the tag and the target protein. Typically, a small oligopeptide ‘stub’ C-terminal to the cleavage site remains attached to the target protein due to the requirements of sequence-specific proteases. Furthermore, steric hindrance can also limit protease efficiency. Here, we show that respec- tively fusing the interacting ePDZ-b/ARVCF protein-peptide pair to the target protein and a protease enables efficient processing of a minimised sequence comprising only residues N-terminal to the cleavage site. Interaction of the protein-peptide pair enforces proximity of the protease and its minimised cleavage sequence, enhancing both catalysis of a sub-optimal site and overcoming steric hindrance. This facilitates the high yield purification of fully native target proteins without recourse to specialised pu- rification columns.
This research was funded by: Agency for Science, Technology and Research Singapore (12302FG015), core funding from IMCB and Young Investigator Grant YIG (A*STAR), NMRC MS-CETSA platform grant MOHIAFCAT2/004/2015 to RMS.