Purification of Escherichia coli alkaline phosphatase on an ion-exchange high-performance liquid chromatographic column using carboxymethyl dextrans.
Carboxymethyl dextrans (CM-Ds) were used on an HPLC ion-exchange column to obtain significantly enriched alkaline phosphatase (EC 3.1.3.1) from a sample of Escherichia coli periplasmic space proteins without significant loss of enzymatic activity. The ability of CM-Ds to separate alkaline phosphatase even when the column was 80-85% saturated with protein demonstrates the potential for high column capacity using CM-Ds. In addition, the fractions containing alkaline phosphatase and CM-Ds were reapplied to the same ion-exchange column under different buffer conditions and purified to homogeneity by salt gradient elution chromatography, thus demonstrating the compatibility of CM-Ds with the latter chromatographic method. The two-step chromatographic procedure yielded enzyme of purity comparable to that of electrophoretically purified E. coli alkaline phosphatase obtained commercially. These studies demonstrate that HPLC displacement chromatography is a mild procedure which allows rapid, quantitative purification of an enzyme. Scaling up with larger columns should allow purification of enzymes of a commercial basis.
D000469:Alkaline Phosphatase / Q000302:isolation & purification*; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003911:Dextrans; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology*; D009696:Nucleic Acids / Q000302:isolation & purification
D003911:Dextrans; D009696:Nucleic Acids; C014392:carboxymethyl dextran; D000469:Alkaline Phosphatase