CLONING AND EXPRESSION OF PURINE NUCLEOSIDE PHOSPHORYLASE-I GENE FROMBACILLUS-STEAROTHERMOPHILUS TH-6-2

Bacillus stearothermophilus TH 6-2 has two kinds of purine nucleoside phosphorylases (Pu-NPase I and Pu-NPase II). The Pu-NPase I is a funct ional homolog of eukaryotic purine nucleoside phosphorylases that can catalyze the phosphorolysis of inosine and guanosine, but not adenosin e, the primary substrate of Pu-NPase II. The Pu-NPase I gene of TH 6-2 has been cloned, sequenced, and expressed in E. coli. The gene corres ponded to an open reading frame of 822 nucleotides that translates int o a putative 274-amino acid protein with a molecular weight of 29,637. The deduced amino terminus sequence completely coincided with that fo und for the purified enzyme, The cloned gene was overexpressed in E. c oli by using the trc promoter to produce an active enzyme in large qua ntities. The amino acid sequence of Pu-NPase I shared 50% similarity w ith those of human and mouse purine nucleoside phosphorylases.