PURIFICATION AND CHARACTERIZATION OF RECOMBINANT THERMOTOGA-MARITIMA DIHYDROFOLATE-REDUCTASE

We have overexpressed the gene for dihydrofolate reductase (DHFR) from Thermotoga maritima in Escherichia coli and characterized the biochem ical properties of the recombinant protein. This enzyme is involved in the ne novo synthesis of deoxythymidine 5'-phosphate and is critical for cell growth. High levels of T. maritima DHFR in the new expression system conferred resistance to high levels of DHFR inhibitors which i nhibit the growth of non-recombinant cells. The enzyme was purified to homogeneity in the following two steps: heat treatment followed by af finity chromatography or cation-exchange chromatography. Most of the b iochemical properties of T. maritima DHFR resemble those of other bact erial or eukaryotic DHFRs, however, some are unique to T. maritima DHF R, The pH optima for activity, K-m for substrates, and polypeptide cha in length of T. maritima DHFR are similar to those of other DHFRs. In addition, the secondary structure of T. maritima DHFR, as measured by circular dichroism, is similar to that of other DHFRs. Interestingly, T. maritima DHFR exhibits some characteristics of eukaryotic DHFRs, su ch as a basic pI, an excess of positively charged residues in the poly peptide chain and activation of the enzyme by inorganic salts and urea . Unlike most other DHFRs which are monomeric or part of a bifunctiona l DHFR-thymidylate synthase (TS) enzyme, T. maritima DHFR seems to gen erally form a dimer in solution and is also much more thermostable tha n other DHFRs. It may be that dimer formation is a key factor in deter mining the stability of T. maritima DHFR.