High-Level Expression of Escherichia coli and Bacillus subtilis Thymidylate Synthases 1 Li-Ming Changchien, Araik Garibian, Verna Frasca, Angelo Lobo, Gladys F. Maley, and Frank Maley 2 New York State Department of Health, Wadsworth Center, P.O. Box 509, Empire State Plaza, Albany, New York 12201-0509 Received February 4, 2000, and in revised form March 20, 2000 Procedures are described for the preparation of highly purified thymidylate synthases from Escherichia coli and Bacillus subtilis. The yields in each case are quite high with about 350 mg of pure protein obtained from 1 liter of cells. Basically all that is required to obtain pure enzyme is an induction step from a high- expression vector, followed by a DE-52 column elution. Both enzymes appeared to be fairly stable in that incu- bation at 43°C for 10 min resulted in the loss of 50% of the E. coli thymidylate synthase activity, while 50°C for 10 min was required to obtain the same effect with the B. subtilis enzyme. In the presence of the substrate, dUMP, each protein was stabilized further by 6 to 7°C, which was increased to 9 to 10°C on addition of dihydrofolate. It was shown also that the E. coli thymidylate synthase could be maintained at 4°C for at least 4 months with little or no loss in activity provided that mercaptoetha- nol was not present. The presence of the latter led to a progressive loss in activity until little activity could be detected after 18 weeks, which was due, in part, to the formation of a disulfide bond with the active site cys- teine. Addition of dithiothreitol restored the enzyme ac- tivity to its original state. © 2000 Academic Press Thymidylate synthase (TS 3 ; EC 2.1.1.45) is the ma- jor provider of dTMP in most cell types and because of its essential role in DNA synthesis this enzyme has become an important target in the assault on bacterial, parasitic, and neoplastic diseases (1). It is therefore of some advantage to have at one’s disposal methods to prepare large quantities of TS from various sources mainly for X-ray diffraction studies, which have be- come an important tool for the rational design of inhib- itors of this enzyme (2). Although much is known about the mechanism of action of this unique enzyme, which is one of the most conserved in nature (1), it has not yet yielded all of its secrets. In particular, potential inter- mediates involved in the final hydride transfer step from tetrahydrofolate to methylene to yield the methyl group of dTMP have been proposed (3–5), but indisput- able verification is still to be provided. Another area that is somewhat murky is how specific inactive mu- tants of Escherichia coli TS can exchange their sub- units to yield a completely revitalized enzyme (6), in support of earlier evidence suggesting that TS is a half-the-sites of activity enzyme. The mechanism by which this exchange occurs is currently under investi- gation and will undoubtedly require considerable amounts of mutant proteins. We and others have described procedures for the isolation of reasonable amounts of TS from E. coli, Lactobacillus casei, L. lactis, Candida albicans, Pneu- mocystis carinii, Cryptococcus neoformans, T4-phage, Varicella zoster, mouse (1), rat (7), and the human (1,8). In this paper we present improvements in the preparation of E. coli and Bacillus subtilis TS, which enable the isolation of hundreds of mg of pure enzyme from 1 liter of cells. In addition we shall describe some unique properties of these enzymes. MATERIALS AND METHODS Growth conditions for overexpression of E. coli and B. subitilis TS. For the overexpression of E. coli TS, 1 liter of TBYE containing 100 g/ml of ampicillin in a 2800-ml Fernbach flask was inoculated with an over- 1 This work was supported in part by Grants CA 44355 from the National Cancer Institute, UPSPHS (F.M.), and MCB-9316321 from the National Science Foundation (G.F.M.). 2 To whom correspondence should be addressed. Fax: (518) 473- 2900. E-mail: maley@wadsworth.org. 3 Abbreviations used: TS, thymidylate synthase; SDS–PAGE, so- dium dodecyl sulfate–polyacrylamide gel electrophoresis; IPTG, iso- propyl -D-thiogalactoside; MSH, mercaptoethanol; DTT, dithiothre- itol; dUMP, 2'-deoxyuridine 5'-phosphate; dTMP, 2'-deoxythymidine 5'-monophosphate; FH 2 7,8-dihydrofolate; TBYE, tryptone broth yeast extract. Protein Expression and Purification 19, 265–270 (2000) doi:10.1006/prep.2000.1245, available online at http://www.idealibrary.com on 265 1046-5928/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.