CDNA CLONING, OVEREXPRESSION IN ESCHERICHIA-COLI, PURIFICATION AND CHARACTERIZATION OF SHEEP LIVER CYTOSOLIC SERINE HYDROXYMETHYLTRANSFERASE

A sheep liver cDNA clone for the cytosolic serine hydroxymethyltransfe rase (SHMT) was isolated and its nucleotide sequence determined. The f ull-length cDNA of SHMT was placed under the control of T7 promoter in pET-3C plasmid and expressed in Escherichia coli. The overexpressed e nzyme, present predominantly in the soluble fraction, was catalyticall y active. The recombinant SHMT was purified to homogeneity with a yiel d of 10 mg/l bacterial culture. The recombinant enzyme was capable of carrying out tetrahydrofolate-dependent and tetrahydrofolate-independe nt reactions as effectively as the native enzyme. The K-m values for s erine (1 mM) and tetrahydrofolate (0.82 mM) were similar to those of t he native enzyme. The recombinant enzyme had a characteristic visible spectrum indicative of the presence of pyridoxal 5'-phosphate as an in ternal aldimine. The apoenzyme obtained upon removal of the cofactor w as inactive and could be reconstituted by the addition of pyridoxal 5' -phosphate demonstrating that the recombinant SHMT was functionally ve ry similar to the native SHMT. This overexpression of eukaryotic tetra meric SHMT in E. coli and the purification and characterization of the recombinant enzyme should thus allow studies on the role of specific amino acids and domains in the activity of the enzyme.