Going native: Complete removal of protein purification affinity tags by simple modification of existing tags and proteases.
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 respectively 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 purification columns.
p53Lab, Agency for Science Technology and Research (A*STAR), 8A Biomedical Grove, 138648, Singapore.
Hui Chin
HC
Goh
Systems Structural Biology Group, Institute of Molecular and Cellular Biology (A*STAR), 61 Biopolis Dr, 138673, Singapore.
Radoslaw M
RM
Sobota
p53Lab, Agency for Science Technology and Research (A*STAR), 8A Biomedical Grove, 138648, Singapore. Electronic address: fghadessy@p53Lab.a-star.edu.sg.
Farid J
FJ
Ghadessy
p53Lab, Agency for Science Technology and Research (A*STAR), 8A Biomedical Grove, 138648, Singapore. Electronic address: saurabh.nirantar79@gmail.com.
Saurabh
S
Nirantar
D002846:Chromatography, Affinity / Q000379:methods; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D010447:Peptide Hydrolases / Q000737:chemistry*; D000072417:Protein Domains; D059748:Proteolysis; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
D011993:Recombinant Fusion Proteins; D010447:Peptide Hydrolases
Affinity tags; Enforced colocalisation; Native protein; Protease; Protein purification