title: Choline-binding domain as a novel affinity tag for purification of fusion proteins produced in Pichia pastoris.
abstract: The choline-binding domain (ChoBD) of the carboxy-terminal region of the Streptococcus pneumoniae amidase LYTA (C-LYTA) presents a strong affinity for tertiary amines. We report a method for single-step purification of proteins expressed in the methylotrophic yeast Pichia pastoris based on the fusion of C-LYTA to the protein of interest. We show that C-LYTA can be efficiently expressed and secreted in this host. Tagged proteins fused to this binding domain can be purified on inexpensive DEAE matrices. It therefore provides a useful system for the purification of recombinant proteins with high specificity suitable for industrial purposes.
authors.affiliation: Departamento de Microbiología II, Facultad de Farmacia, Universidad Complutense, Plaza de Ramón y Cajal s/n. 28040, Madrid, Spain.
authors.forename: J
authors.initials: J
authors.lastname: Caubín
authors.forename: H
authors.initials: H
authors.lastname: Martín
authors.forename: A
authors.initials: A
authors.lastname: Roa
authors.forename: I
authors.initials: I
authors.lastname: Cosano
authors.forename: M
authors.initials: M
authors.lastname: Pozuelo
authors.forename: J M
authors.initials: JM
authors.lastname: de La Fuente
authors.forename: J M
authors.initials: JM
authors.lastname: Sánchez-Puelles
authors.forename: M
authors.initials: M
authors.lastname: Molina
authors.forename: C
authors.initials: C
authors.lastname: Nombela
mesh_terms: D001483:Base Sequence; D001665:Binding Sites; D002794:Choline / Q000378:metabolism*; D002848:Chromatography, DEAE-Cellulose / Q000379:methods*; D008969:Molecular Sequence Data; D009238:N-Acetylmuramoyl-L-alanine Amidase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D013296:Streptococcus pneumoniae / Q000201:enzymology / Q000235:genetics; D001618:beta-Lactamases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D011993:Recombinant Fusion Proteins; C076332:EJL amidase; D009238:N-Acetylmuramoyl-L-alanine Amidase; D001618:beta-Lactamases; D002794:Choline