Construction and characterization of a single polypeptide chain containing two enzymatically active dihydrofolate reductase domains.
A single polypeptide chain containing two dihydrofolate reductase (DHFR) sequences from Escherichia coli was constructed to determine if a repeat sequence fusion protein could be expressed in an active form. The possibility that intersequence interactions could play a significant role for this enzyme is suggested by the results of Hall and Frieden (1989, Proc. Natl Acad. Sci. USA, 86, 3060-3064) who observed a substantial decrease in the yield of active enzyme when folded in the presence of a large C-terminal fragment. The fusion protein [DHFR(Cys152Glu)--Ile--DHFR (Met1Gln)] was efficiently expressed in E. coli cells and has an activity which is twice that of the wild-type enzyme in the standard assay. The Michaelis constants of the fusion protein for the substrate, dihydrofolate and the cofactor, NADPH, are essentially unchanged from those of the wild-type protein. The urea-induced in vitro unfolding reaction of the fusion protein at low concentrations was found to be fully reversible and follow a three state model, suggesting that the two domains unfold independently. At higher protein concentrations the unfolding transition broadened and shifted to a higher urea concentration. Size-exclusion chromatography results are consistent with the formation of aggregates at the higher protein concentration, even in the absence of denaturant.
Iwakura
M
M
Research Institute for Polymers and Textiles, Ibaraki, Japan.
Matthews
C R
CR
D000595:Amino Acid Sequence; D001483:Base Sequence; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology*; D005818:Genetic Engineering; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009154:Mutation; D011487:Protein Conformation; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012091:Repetitive Sequences, Nucleic Acid / Q000235:genetics; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics* / Q000378:metabolism
D011993:Recombinant Fusion Proteins; D013762:Tetrahydrofolate Dehydrogenase