Choline-binding domain as a novel affinity tag for purification of fusion proteins produced in Pichia pastoris.
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.
Caubín
J
J
Departamento de Microbiología II, Facultad de Farmacia, Universidad Complutense, Plaza de Ramón y Cajal s/n. 28040, Madrid, Spain.
Martín
H
H
Roa
A
A
Cosano
I
I
Pozuelo
M
M
de La Fuente
J M
JM
Sánchez-Puelles
J M
JM
Molina
M
M
Nombela
C
C
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
D011993:Recombinant Fusion Proteins; C076332:EJL amidase; D009238:N-Acetylmuramoyl-L-alanine Amidase; D001618:beta-Lactamases; D002794:Choline