RECOMBINANT URACIL PHOSPHORIBOSYLTRANSFERASE FROM THE THERMOPHILE BACILLUS-CALDOLYTICUS - EXPRESSION, PURIFICATION, AND PARTIAL CHARACTERIZATION

The upp gene encoding the major uracil phosphoribosyltransferase (UPRT ) of the thermophile Bacillus caldolyticus was cloned by complementati on of an Escherichia coli upp mutation. The nucleotide sequence of the cloned DNA revealed all open reading frame of 630 bp encoding a polyp eptide of 209 amino acids (M-r 22,817) with 84% amino acid sequence id entity to the deduced upp gene product, of Bacillus subtilis. Primer e xtension analysis indicated that the transcriptional start site of the cloned gene was positioned 37 or 38 bp upstream of the coding region. When overexpressed in E. coli, the recombinant UPRT represented appro ximately 18% of the soluble cellular proteins. The enzyme was purified to homogeneity by two sequential precipitations with 50 mM Na-phospha te, pH 7.0. Gel filtration chromatography indicated that the native en zyme existed as a dimer at high protein concentrations but that it dis sociated to a monomeric form on dilution, In dilute solutions the enzy me is highly unstable but can be stabilized by addition of bovine seru m albumin. In concentrated solution (>5 mg/ml) the enzyme is stable fa r months at 4 degrees C, even in the absence of bovine serum albumin. By comparing the UPRT activity of crude extracts of B. subtilis and B. caldolyticus it was found that the enzyme from B. caldolyticus was co nsiderably more stable toward thermal inactivation than the homologous enzyme from B. subtilis. (C) 1997 Academic Press.