CLONING AND CHARACTERIZATION OF A THERMOSTABLE ALPHA-DNA POLYMERASE FROM THE HYPERTHERMOPHILIC ARCHAEON THERMOCOCCUS SP. TY

The gene for an extremely thermostable DNA polymerase has been cloned from chromosomal DNA of the recently characterised hyperthermophilic a rchaeon Thermococcus sp. TY by using degenerate primers derived from c onsensus 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 eleme nts, so-called ''inteins''. This is the first DNA polymerase containin g such a large number of self-splicing elements. To ensure an efficien t expression, these regions were deleted on the DNA level. The resulti ng protein showed DNA polymerase and 3'-5' exonuclease activity at hig h temperatures, being a promising candidate for use in the polymerase chain reaction (PCR). (C) 1997 Elsevier Science B.V.