Cloning and expression of the inorganic pyrophosphatase gene from thermophilic bacterium PS-3

The thermophilic bacterium PS-3 ppa gene encoding inorganic pyrophosphatase (PPiase) has been cloned and sequenced. The deduced amino acid sequence was exactly the same as that determined on Edman degradation (Ichiba, T., Takenaka, O., Samejima, T. and Hachimori, A., J. Biochem. 108: 572-578, 1990) except that amino acid-70 is threonine instead of proline and two more amino acids, Asn-Lys, are present at the carboxyl terminus. The structural gene containing BamH I and Hind III restriction cleaving sites at the 5' and 3' ends, respectively, was amplified by the PCR method. Escherichia coli JM109 was transformed with a synthetic DNA, and we found that thermophilic ppa was expressed in E. coli, as judged on enzyme assaying, SDS-PAGE and immune assaying.