title: Single-column purification of free recombinant proteins using a self-cleavable affinity tag derived from a protein splicing element.
abstract: A novel protein purification system has been developed which enables purification of free recombinant proteins in a single chromatographic step. The system utilizes a modified protein splicing element (intein) from Saccharomyces cerevisiae (Sce VMA intein) in conjunction with a chitin-binding domain (CBD) from Bacillus circulans as an affinity tag. The concept is based on the observation that the modified Sce VMA intein can be induced to undergo a self-cleavage reaction at its N-terminal peptide linkage by 1,4-dithiothreitol (DTT), beta-mercaptoethanol (beta-ME) or cysteine at low temperatures and over a broad pH range. A target protein is cloned in-frame with the N-terminus of the intein-CBD fusion, and the stable fusion protein is purified by adsorption onto a chitin column. The immobilized fusion protein is then induced to undergo self-cleavage under mild conditions, resulting in the release of the target protein while the intein-CBD fusion remains bound to the column. No exogenous proteolytic cleavage is needed. Furthermore, using this procedure, the purified free target protein can be specifically labeled at its C-terminus.
authors.affiliation: New England Biolabs, Beverly, MA 01915, USA.
authors.forename: S
authors.initials: S
authors.lastname: Chong
authors.forename: F B
authors.initials: FB
authors.lastname: Mersha
authors.forename: D G
authors.initials: DG
authors.lastname: Comb
authors.forename: M E
authors.initials: ME
authors.lastname: Scott
authors.forename: D
authors.initials: D
authors.lastname: Landry
authors.forename: L M
authors.initials: LM
authors.lastname: Vence
authors.forename: F B
authors.initials: FB
authors.lastname: Perler
authors.forename: J
authors.initials: J
authors.lastname: Benner
authors.forename: R B
authors.initials: RB
authors.lastname: Kucera
authors.forename: C A
authors.initials: CA
authors.lastname: Hirvonen
authors.forename: J J
authors.initials: JJ
authors.lastname: Pelletier
authors.forename: H
authors.initials: H
authors.lastname: Paulus
authors.forename: M Q
authors.initials: MQ
authors.lastname: Xu
mesh_terms: D000345:Affinity Labels; D000595:Amino Acid Sequence; D001483:Base Sequence; D002352:Carrier Proteins / Q000737:chemistry; D002686:Chitin; D005822:Genetic Vectors; D006863:Hydrogen-Ion Concentration; D058619:Maltose-Binding Proteins; D008722:Methods; D008969:Molecular Sequence Data; D011499:Protein Processing, Post-Translational; D019154:Protein Splicing; D006180:Proton-Translocating ATPases / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification*; D013696:Temperature; D025262:Vacuolar Proton-Translocating ATPases
chemical_list: D000345:Affinity Labels; D002352:Carrier Proteins; D058619:Maltose-Binding Proteins; D011994:Recombinant Proteins; D002686:Chitin; D025262:Vacuolar Proton-Translocating ATPases; D006180:Proton-Translocating ATPases