EXPRESSION IN ESCHERICHIA-COLI OF THE THERMOSTABLE DNA-POLYMERASE FROM PYROCOCCUS-FURIOSUS

Pfu, the DNA polymerase from Pyrococcus furiosus, has the lowest error rate of any known polymerase in polymerase chain reaction (PCR) ampli fication, Previously the protein has been purified from P. furiosus ba cterial cultures, and a recombinant form has been produced in a baculo virus system. We have produced a pET plasmid for expression of Pfu in Escherichia coli (the expression plasmid pETpfu is available from ATCC , Accession No. 87496) and found that this plasmid is toxic or unstabl e in the expressing strain BL21(DE3), even in the absence of induction . However, the plasmid was stable in BL21(DE3) containing the pLysS pl asmid, which suppresses expression prior to induction, and a 90-kDa pr otein was expressed upon addition of isopropyl beta-D-thiogalactopyran oside. The protein was purified by heating (to denature E. coli protei ns), followed by chromatography on P11 phosphocellulose and mono Q col umns. The purified protein had the same activity as the commercially o btained baculovirus-expressed Pfu in both DNA polymerase and PCR react ions. This bacterial expression system appears to be the method of cho ice for production of Pfu. (C) 1997 Academic Press.