Rapid large-scale preparation of recombinant Erwinia chrysanthemi L-asparaginase

L-asparaginase from Erwinia provides an alternative to the enzyme from E. coli for the effective treatment of acute lymphoblastic leukaemia. A procedure was required for the large-scale partial purification of the recombinant Erwinia enzyme cloned and expressed in Erwinia. Enzyme was extracted from Erwinia at high pH and extraneous protein precipitated at low pH. S-Sepharose FF was selected as the medium of choice for the chromatography step since it was adequate for the high flow rates required (linear flow rate 315 cm h-1) and the methylsulphonate functional groups exploited the high pI of the enzyme by allowing binding of L-asparaginase at pH 4.8 while most of the other proteins passed through the column. The useful capacity of the matrix was up to 34 mg enzyme/ml matrix at a linear flow rate of 95 cm h-1 and 15.4 mg enzyme/ml matrix at a linear flow rate of 315 cm h-1. Weakly bound protein was removed by a wash at pH 6.0. The L-asparaginase was eluted by a wash at pH 6.8 (linear flow rate 95 cm h-1) and was substantially pure, only requiring polishing steps to be suitable for use as a parenteral agent. The purity of the protein was complemented by a 92% recovery of active enzyme from this cation-exchange matrix.