Cloning and characterisation of a thermostable alpha-DNA polymerase from the hyperthermophilic archaeon Thermococcus...

The gene for an extremely thermostable DNA polymerase has been cloned from chromosomal DNA of the recently characterised hyperthermophilic archaeon Thermococcus sp. TY by using degenerate primers derived from consensus sequences of known archaeal enzymes. The corresponding enzyme was overexpressed in Escherichia coli. Sequence comparison of the gene with related DNA polymerase genes revealed that it is interrupted by three regions showing high similarities to self-splicing protein elements, so-called "inteins". This is the first DNA polymerase containing such a large number of self-splicing elements. To ensure an efficient expression, these regions were deleted on the DNA level. The resulting protein showed DNA polymerase and 3'-5' exonuclease activity at high temperatures, being a promising candidate for use in the polymerase chain reaction (PCR).