EXPRESSION, PURIFICATION, AND CHARACTERIZATION OF THYMIDYLATE SYNTHASE FROM LACTOCOCCUS-LACTIS

The thymidylate synthase (TS) gene from Lactococcus lactis has been hi ghly expressed in Escherichia coli. The TS protein was purified by seq uential chromatography on Q-Sepharose and phenyl-Sepharose. Six grams of cell pellet yielded 140 mg of homogeneous TS. TS is a highly conser ved enzyme, and several of the conserved amino acid residues that have been implicated in catalytic function are altered in L. lactis TS. By use of a 3-dimensional homology model, we have predicted covariant ch anges that might compensate for these differences. With the large amou nts of L. lactis TS now available, studies can be pursued to understan d the structure-function relationships of this enzyme compared to othe r TSs and to confirm the presumed roles of the compensatory changes pr edicted in the homology model.