Abstract
The Escherichia coli gene for folylpolyglutamate synthetase-dihydrofolate synthetase was localized to plasmids pLC22-45, 24-31, and 28-44 of the Clarke-Carbon E. coli colony bank (Clarke, L., and Carbon, J. (1976) Cell 9, 91-99) by screening the bank by replica mating with an E. coli folC mutant. The folC gene was subcloned from pLC22-45 and inserted into a high copy number plasmid containing the lambda replication control region under the control of the temperature-sensitive cI857 repressor and into a high expression plasmid containing the lambda PL promoter and the cI857 repressor. The folC structural gene was located on a 1.52-kilobase PvuI fragment, sufficient to code for a protein of maximum Mr 55,000. E. coli transformants containing the recombinant plasmids, when induced by culturing at 42 degrees C, had folylpolyglutamate synthetase and dihydrofolate synthetase levels that were 100- to 400-fold higher than in wild type strains and which represented up to 4% of the soluble cell protein. The E. coli folylpolyglutamate synthetase-dihydrofolate synthetase has been purified to homogeneity from the transformants. Both activities are catalyzed by a single protein of Mr 47,000. Some kinetic properties of the enzymes and a new spectrophotometric method for assaying dihydrofolate synthetase activity are described.
Highlights
CLONING AND HIGH EXPRESSION OF THE ESCHERICHICAOLI folC GENE AND PURIFICATION AND PROPERTIES OF THE GENE PRODUCT*
The Corynebacterium and E. coli proteins alsopossessdihydrofolate synthetase activity, and preliminary studieshave suggested that thetwo synthetase activities are catalyzed by separate sites and that the proteisinbifunctional [5, 9,10,11]
The protein has beenpurified to homogeneity from Lactobacillus [7] and Corynebacterium [5] and partially purified from E . coli R [8, 9] andsome of its properties characterized
Summary
Transformants that grew onminimal medium plates plus ampicillin contained a plasmid (pBC10-4, 11.2 kb, Fig. 1)with a 5.5-kb BgZII/ClaI insert and had elevated enzyme levels (Table I). The fragment was restricted with SstII, digest was ligated to HindIII-treated pKC7, and the mixture and a 3.5-kb fragment was isolated and blunt endedwith T4 was used to transform SF2 to growth on minimal medium polymerase. Enzyme lev- ants indicated that the two classes of transformants correels in these transformants were at wild type levels or lower, sponded toopposite orientations of the SstII fragmentin the higher than in SF2 (Table I).
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