Rapid and efficient tag removal remains a significant problem in recombinant protein purification. Using an engineered DnaE intein from Nostoc punctiforme, we developed a split intein mediated ultra-rapid purification (SIRP) method for the purification of tagless recombinant protein from E. coli lysate in less than 1 h. This system exhibits extraordinarily rapid thio-induced C-terminal cleavage with about 50% completion within 30 s at both 22°C and 6°C. This is the fastest C-terminal cleavage activity reported to date for inteins. Although the reaction kinetics slow down after the first minute, >90% cleavage completion is achieved within 30 min at 22°C, or within 3 h at 6°C. The ultra-rapid cleavage kinetics are made possible by the positioning of the purification tag at the split junction to the C-terminus of the intein N-fragment, thus avoiding potential steric hindrance of the critical interaction between the N- and C-extein. Target proteins are cleaved to >72% completion after 1 h of intein reaction regardless of the identity of the N-terminal amino acid except in the cases of threonine (50% cleavage) and proline. 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. Biotechnol. Bioeng. 2013; 110:2471–2481. © 2013 Wiley Periodicals, Inc.