Split intein mediated ultra‐rapid purification of tagless protein (SIRP). Issue 9 (22nd April 2013)

• Record Type: Journal Article
• Authors: Guan, Dongli; Ramirez, Miguel; Chen, Zhilei
• Is Part Of: Biotechnology and bioengineering. Volume 110:Issue 9(2013:Sep.)
• Page Start: 2471
• Page End: 2481
• Publication Date: 2013-04-22
• Subjects: Biotechnology -- Periodicals; Bioengineering -- Periodicals; 660.6
• DOI: 10.1002/bit.24913
• Languages: English
• ISSNs: 0006-3592

Abstract

<abstract abstract-type="main" xml:lang="en"> <title>ABSTRACT</title> <sec id="bit24913-sec-0001" sec-type="section"> <p>Rapid and efficient tag removal remains a significant problem in recombinant protein purification. Using an engineered DnaE intein from <italic>Nostoc punctiforme</italic>, we developed a split intein mediated ultra‐rapid purification (SIRP) method for the purification of tagless recombinant protein from <italic>E. coli</italic> 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<sup>2+</sup> 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.</p> </sec> </abstract>