Dihydrofolate reductase from Bacillus subtilis and its artificial derivatives: expression, purification, and characterization.
The Bacillus subtilis dihydrofolate reductase (DHFR) gene was expressed in Escherichia coli. The gene product was purified to homogeneity by Butyl-Toyopearl, Toyopearl HW55, and DEAE-Toyopearl column chromatographies, and its molecular properties were compared to those of E. coli DHFR. The specific enzyme activity of the B. subtilis DHFR was 240 units/mg under the standard assay conditions, being about four times higher than that of the E. coli DHFR. Km for coenzyme NADPH was 20.7 microM, a value about three times larger than that of E. coli, whereas Km (1.5 microM) for the substrate, dihydrofolate, was similar to that of E. coli DHFR. This seems to reflect the low homology of the amino acid sequence in residues 61-88 of the two DHFRs where one of the NADPH binding sites is located [Bystrof, C. & Kraut, J. (1991) Biochemistry 30, 2227-2239]. Similar to the E. coli DHFR [Iwakura, M. et al. (1992) J. Biochem. 111, 37-45], the extension of amino acid sequences at the C-terminal end of the B. subtilis DHFR could be attained without loss of the enzyme function or decrease of the protein yield. Thus, the DHFR is useful as a carrier protein for expressing small polypeptides, such as leucine enkephalin, bradykinin, and somatostatin.
Iwakura
M
M
Research Institute for Polymers and Textiles, Ibaraki.
Tanaka
T
T
D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics*; D001483:Base Sequence; D001665:Binding Sites; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D007700:Kinetics; D008969:Molecular Sequence Data; D009249:NADP / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism
D004269:DNA, Bacterial; D011993:Recombinant Fusion Proteins; D009249:NADP; D013762:Tetrahydrofolate Dehydrogenase