Cloning and expression of the gene encoding Lactobacillus casei folylpoly-gamma-glutamate synthetase in Escherichia coli and determination of its primary structure.
A genomic library of Lactobacillus casei DNA containing 10,000 individual clones was constructed in the plasmid pUC13. The gene encoding the L. casei folylpolyglutamate synthetase was isolated from the library by complementation of a folC mutant of Escherichia coli. The gene was expressed in E. coli from its own promoter and produced amplified folylpolyglutamate synthetase activity with properties identical with those of the purified L. casei enzyme. The absence of dihydrofolate synthetase activity and the preferential utilization of 5,10-methylenetetrahydrofolate, rather than 10-formyltetrahydrofolate as folate substrate, distinguishes this activity from the E. coli folylpolyglutamate synthetase-dihydrofolate synthetase. A protein of Mr = 43,000, identical with that of purified L. casei folylpolyglutamate synthetase, was expressed in maxicells containing the complementing plasmid. The nucleotide sequence of the folylpolyglutamate synthetase gene was determined. An open reading frame of 1,284 bases was found predicting a protein product of 428 amino acids with Mr = 44,169. The predicted amino acid sequence of the gene is 33% homologous to that of the E. coli folylpolyglutamate synthetase. Primer extension studies indicate that the transcription initiation site is at -59 base pairs, relative to the initiation ATG codon of the folylpolyglutamate synthetase gene, suggesting that the gene is transcribed independently of upstream genes. A second open reading frame was found downstream of the folylpolyglutamate synthetase open reading frame, overlapping the final codon by 1 base pair. This downstream gene may be co-transcribed with the folylpolyglutamate synthetase gene.
Toy
J
J
Department of Microbiology, University of Toronto, Ontario, Canada.
Bognar
A L
AL
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics*; D005798:Genes, Bacterial; D015698:Genomic Library; D007780:Lacticaseibacillus casei / Q000201:enzymology / Q000235:genetics*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010453:Peptide Synthases / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids; D011994:Recombinant Proteins / Q000378:metabolism; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid; D012984:Software
D004269:DNA, Bacterial; D011994:Recombinant Proteins; D010453:Peptide Synthases; C023103:folylpolyglutamate synthetase