Folylpoly-gamma-glutamate synthetase-dihydrofolate synthetase. Cloning and high expression of the Escherichia coli folC gene and purification and properties of the gene product.
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.
Bognar
A L
AL
Osborne
C
C
Shane
B
B
Singer
S C
SC
Ferone
R
R
D003001:Cloning, Molecular; D004262:DNA Restriction Enzymes / Q000378:metabolism; D004269:DNA, Bacterial / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D005786:Gene Expression Regulation; D007700:Kinetics; D008970:Molecular Weight; D009097:Multienzyme Complexes; D010453:Peptide Synthases / Q000235:genetics*; D010957:Plasmids
D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D009097:Multienzyme Complexes; D004262:DNA Restriction Enzymes; D010453:Peptide Synthases; C500101:folC protein, E coli; C020041:dihydrofolate synthetase; C023103:folylpolyglutamate synthetase