title: Affinity Purification of Proteins in Tag-Free Form: Split Intein-Mediated Ultrarapid Purification (SIRP).
abstract: Proteins purified using affinity-based chromatography often exploit a recombinant affinity tag. Existing methods for the removal of the extraneous tag, needed for many applications, suffer from poor efficiency and/or high cost. Here we describe a simple, efficient, and potentially low-cost approach-split intein-mediated ultrarapid purification (SIRP)-for both the purification of the desired tagged protein from Escherichia coli lysate and removal of the tag in less than 1 h. The N- and C-fragment of a self-cleaving variant of a naturally split DnaE intein from Nostoc punctiforme are genetically fused to the N-terminus of an affinity tag and a protein of interest (POI), respectively. The N-intein/affinity tag is used to functionalize an affinity resin. The high affinity between the N- and C-fragment of DnaE intein enables the POI to be purified from the lysate via affinity to the resin, and the intein-mediated C-terminal cleavage reaction causes tagless POI to be released into the flow-through. The intein cleavage reaction is strongly inhibited by divalent ions (e.g., Zn2+) under non-reducing conditions and is significantly enhanced by reducing conditions. The POI is cleaved efficiently regardless of the identity of the N-terminal amino acid except in the cases of threonine and proline, and the N-intein-functionalized affinity resin can be regenerated for multiple cycles of use.
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.affiliation: Department of Chemical Engineering, Texas A&M University, College Station, TX, 77843, USA. zchen4@tamu.edu.|Department of Microbial and Molecular Pathogenesis, Texas A&M University Health Science Center, College Station, TX, 77843, USA. zchen4@tamu.edu.
authors.forename: Zhilei
authors.initials: Z
authors.lastname: Chen
mesh_terms: D002846:Chromatography, Affinity / Q000379:methods*; D004258:DNA Polymerase III / Q000737:chemistry* / Q000235:genetics*; D047668:Inteins; D046937:Nostoc; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
chemical_list: D011993:Recombinant Fusion Proteins; C074904:DNA polymerase III, alpha subunit; D004258:DNA Polymerase III
keywords: Chitin-binding domain; Chromatography; Protein purification; Tag removal; Tagless