title: Split intein mediated ultra-rapid purification of tagless protein (SIRP).
abstract: 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 Zn(2+) 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.
authors.affiliation: Department of Chemical Engineering, Texas A&M University, College Station, TX 77843, USA.
authors.forename: Dongli
authors.initials: D
authors.lastname: Guan
authors.forename: Miguel
authors.initials: M
authors.lastname: Ramirez
authors.forename: Zhilei
authors.initials: Z
authors.lastname: Chen
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics; D002686:Chitin / Q000737:chemistry / Q000378:metabolism; D002846:Chromatography, Affinity / Q000379:methods; D004258:DNA Polymerase III / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D047668:Inteins / Q000235:genetics*; D007700:Kinetics; D008958:Models, Molecular; D046937:Nostoc / Q000235:genetics; D011485:Protein Binding; D015202:Protein Engineering / Q000379:methods*; D019154:Protein Splicing; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D013997:Time Factors
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D002686:Chitin; C074904:DNA polymerase III, alpha subunit; D004258:DNA Polymerase III
keywords: C-terminal cleavage; Nostoc; chitin-binding domain; inteins; kinetics; protein engineering; split intein