Generation of an affinity column for antibody purification by intein-mediated protein ligation.
Coupling an antigenic peptide to a solid support is a crucial step in the affinity purification of a peptide-specific antibody. Conventional methods for generating reactive agarose, cellulose or other matrices for peptide conjugation are laborious and can result in a significant amount of chemical waste. In this report, we present a novel method for the facile production of a peptide affinity column by employing intein-mediated protein ligation (IPL) in conjunction with chitin affinity chromatography. A reactive thioester was generated at the C-terminal of the chitin binding domain (CBD) from the chitinase A1 of Bacillus circulans WL-2 by thiol-induced cleavage of the peptide bond between the CBD and a modified intein. Peptide epitopes possessing an N-terminal cysteine were ligated to the chitin bound CBD tag. We demonstrate that the resulting peptide columns permit the highly specific and efficient affinity purification of antibodies from animal sera.
Sun
Luo
L
New England BioLabs, Inc, 32 Tozer Road, Beverly, MA 01915, USA.
Ghosh
Inca
I
Xu
Ming-Qun
MQ
D000595:Amino Acid Sequence; D000906:Antibodies / Q000302:isolation & purification*; D000918:Antibody Specificity; D001665:Binding Sites; D002686:Chitin / Q000378:metabolism; D002846:Chromatography, Affinity; D008969:Molecular Sequence Data
D000906:Antibodies; D002686:Chitin