METHOD FOR PURIFYING PROKARYOTIC PHENYLALANINE AMMONIA-LYASE VARIANTS

摘要

The present invention provides a method for purifying an Anabaena variabilis phenylalanine ammonia-lyase (AvPAL) variant with minimal aggregation. The method comprises the steps of (a) lysing bacterial cells containing the AvPAL variant by homogenization to generate a cell lysate; (b) heating the cell lysate to 65°C for 30 to 120 minutes; (c) centrifuging the heated cell lysate, wherein a supernatant comprising the AvPAL variant is retained; (d) filtering the supernatant to remove precipitates; and (e) separating the AvPAL variant from contaminating proteins by sequential chromatography over an anion exchange (AIEX) column followed by a hydrophobic interaction (HIC) column, wherein the eluate from the HIC column comprises the AvPAL variant, wherein the cysteine residues at positions 503 and 565 of said AvPAL variant have been substituted by serine residues (SEQ ID NO:11).