Recombinant Uracil Phosphoribosyltransferase from the ThermophileBacillus caldolyticus:Expression, Purification, and Partial Characterization

Abstract

Theuppgene encoding the major uracil phosphoribosyltransferase (UPRT) of the thermophileBacillus caldolyticuswas cloned by complementation of anEscherichia coli uppmutation. The nucleotide sequence of the cloned DNA revealed an open reading frame of 630 bp encoding a polypeptide of 209 amino acids (Mr22,817) with 84% amino acid sequence identity to the deduceduppgene product ofBacillus subtilis.Primer extension analysis indicated that the transcriptional start site of the cloned gene was positioned 37 or 38 bp upstream of the coding region. When overexpressed inE. coli,the recombinant UPRT represented approximately 18% of the soluble cellular proteins. The enzyme was purified to homogeneity by two sequential precipitations with 50 mmNa-phosphate, pH 7.0. Gel filtration chromatography indicated that the native enzyme existed as a dimer at high protein concentrations but that it dissociated to a monomeric form on dilution. In dilute solutions the enzyme is highly unstable but can be stabilized by addition of bovine serum albumin. In concentrated solution (>5 mg/ml) the enzyme is stable for months at 4°C, even in the absence of bovine serum albumin. By comparing the UPRT activity of crude extracts ofB. subtilisandB. caldolyticusit was found that the enzyme fromB. caldolyticuswas considerably more stable toward thermal inactivation than the homologous enzyme fromB. subtilis.