Rapid and efficient tag removal remains a significant problem in recombinant protein purification. Using an engineered C-terminal cleaving naturally split DnaE intein from Nostoc punctiforme, we developed a recombinant protein purification approach � split intein mediated ultra-rapid purification of tagless protein (SIRP) � that enables the purification of tagless recombinant protein from E. coli lysate in less than 1 hour. In SIRP, the protein purification tag is inserted at the intein split junction (C-terminus of the intein N-fragment) and the target protein is fused to the C-terminus of the C-fragment. This system exhibits extraordinarily rapid thio-induced C-terminal cleavage with about 50% completion within 30 seconds at both 22 �C and 6 �C. This is the fastest C-terminal cleavage activity reported to date for inteins. Although the reaction kinetics appears to slow down after the first few minutes, >85% cleavage completion is achieved within 30 minutes at 22 �C, or within 3 h at 6 �C. The ultra-rapid cleavage kinetics is made possible by the positioning of the purification tag at the intein split junction, thus avoiding potential steric hindrance of the critical interaction between the N- and C-extein. Generally speaking, the C-terminal cleavage efficiency of our engineered split intein was not affected by the identity of the first residue of the C-extein (proline was found to be an exception), enabling SIRP to completely remove the purification tag and purify proteins with the native N termini. The C-terminal cleavage reaction can be effectively inhibited by divalent Zn2+ under non-reducing conditions. Importantly, the association of the intein N- and C-fragments is reversible, enabling the column-bound intein N-fragment bait protein to be regenerated for multiple usages and further reducing the cost of protein purification. SIRP technology should provide a useful tool for the purification of tagless proteins and peptides.

Split intein mediated ultra-rapid purification of tagless protein (SIRP)

245th National Meeting (2013)

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