title: Utilizing the C-terminal cleavage activity of a protein splicing element to purify recombinant proteins in a single chromatographic step.
abstract: A conventional affinity protein purification system often requires a separate protease to separate the target protein from the affinity tag. This paper describes a unique protein purification system in which the target protein is fused to the C-terminus of a modified protein splicing element (intein). A small affinity tag is inserted in a loop region of the endonuclease domain of the intein to allow affinity purification. Specific mutations at the C-terminal splice junction of the intein allow controllable C-terminal peptide bond cleavage. The cleavage is triggered by addition of thiols such as dithiothreitol or free cysteine, resulting in elution of the target protein while the affinity-tagged intein remains immobilized on the affinity column. This system eliminates the need for a separate protease and allows purification of a target protein without the N-terminal methionine. We have constructed general cloning vectors and demonstrated single-column purification of several proteins. In addition, we discuss several factors that may affect the C-terminal peptide bond cleavage activity.
authors.affiliation: New England Biolabs Inc., 32 Tozer Road, Beverly, MA 01915, USA. chong@neb.com
authors.forename: S
authors.initials: S
authors.lastname: Chong
authors.forename: G E
authors.initials: GE
authors.lastname: Montello
authors.forename: A
authors.initials: A
authors.lastname: Zhang
authors.forename: E J
authors.initials: EJ
authors.lastname: Cantor
authors.forename: W
authors.initials: W
authors.lastname: Liao
authors.forename: M Q
authors.initials: MQ
authors.lastname: Xu
authors.forename: J
authors.initials: J
authors.lastname: Benner
mesh_terms: D000345:Affinity Labels; D001483:Base Sequence; D002846:Chromatography, Affinity / Q000379:methods*; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification*
chemical_list: D000345:Affinity Labels; D017931:DNA Primers; D011993:Recombinant Fusion Proteins