Reversed-flow affinity elution applied to the purification of carboxypeptidase Y.
In the present study we describe a novel method for obtaining highly pure carboxypeptidase Y, or derivatives thereof, in a single-step purification procedure. The method is based on affinity chromatography and the results demonstrate that an efficient method is obtained only when the affinity gel is fully saturated with enzyme. Thus, pilot experiments are required to determine the binding capacity of the resin with respect to a given enzyme. To avoid this additional experimental effort, we have developed a method utilizing reversed-flow affinity elution. The method has been successfully employed to purify hundreds of carboxypeptidase Y mutant enzymes.
Mortensen
U H
UH
Carlsberg Laboratory, Department of Chemistry, Copenhagen, Denmark.
Stennicke
H R
HR
Breddam
K
K
D002268:Carboxypeptidases / Q000235:genetics / Q000302:isolation & purification*; D043402:Cathepsin A; D002846:Chromatography, Affinity / Q000379:methods*; D016296:Mutagenesis; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification
D011994:Recombinant Proteins; D002268:Carboxypeptidases; D043402:Cathepsin A