title: Protein Affinity Purification using Intein/Chitin Binding Protein Tags.
abstract: Isolation of highly purified recombinant protein is essential for a wide range of biochemical and biophysical assays. Affinity purification in which a tag is fused to the desired protein and then specifically bound to an affinity column is a widely used method for obtaining protein of high purity. Many of these methods have the drawbacks of either leaving the recombinant tag attached to the protein or requiring the addition of a protease which then must be removed by further chromatographic steps. The fusion of a self-cleaving intein sequence followed by a chitin-binding domain (CBD) allows for one-step chromatographic purification of an untagged protein through the thiol-catalyzed cleavage of the intein sequence from the desired protein. The affinity purification is highly specific and can yield pure protein without any undesired N- or C-terminal extensions. This protocol is based on the IMPACT™-System (intein mediated purification with an affinity chitin-binding tag) marketed by New England Biolabs.
authors.affiliation: Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
authors.forename: Sarah F
authors.initials: SF
authors.lastname: Mitchell
authors.affiliation: Laboratory on the Mechanism and Regulation of Protein Synthesis Eunice Kennedy Shriver National Institute of Child Health and Human Development National Institutes of Health Bethesda, MD, USA. Electronic address: jlorsch@jhmi.edu.
authors.forename: Jon R
authors.initials: JR
authors.lastname: Lorsch
mesh_terms: D001671:Biochemistry / Q000379:methods; D002384:Catalysis; D002686:Chitin / Q000737:chemistry*; D002846:Chromatography, Affinity / Q000295:instrumentation* / Q000379:methods*; D004926:Escherichia coli / Q000378:metabolism; D047668:Inteins; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000302:isolation & purification*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000737:chemistry; D013696:Temperature
chemical_list: D011506:Proteins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D013438:Sulfhydryl Compounds; D002686:Chitin
keywords: CBD; Cell harvesting; Cell lysis; Chitin beads; IMPACT™-System; Intein/Chitin binding protein tags; Protein affinity purification