CLONING, NUCLEOTIDE-SEQUENCE, AND EXPRESSION IN ESCHERICHIA-COLI OF DNA-POLYMERASE GENE (POLA) FROM THERMUS-THERMOPHILUS HB8

A gene coding for a thermostable DNA polymerase I (Tth Pol I) was cloned from Thermus thermophilus HB8, and its nucleotide sequence was identified. The Tth Pol I gene (polA) has an open reading frame of 2505 base pairs available to encode a peptide of 834 amino acids. Another incomplete open reading frame was also found upstream from the polA gene. In the deduced amino acid sequence of Tth Pol I, which shows 87% similarity to that of Thermus aquaticus DNA polymerase, there is the leucine zipper structure from Leu458 to Leu486. The Tth Pol I gene was subcloned in a high-expression vector. Escherichia coli cells harboring the hybrid plasmid produced about 100,000 units of thermostable DNA polymerase in a 200-ml culture.