Characterization of Recombinant Thermococcus kodakaraensis (KOD) DNA Polymerases Produced Using Silkworm-Baculovirus Expression Vector System

The KOD DNA polymerase from Thermococcus kodakarensis (Tkod-Pol) has been preferred for PCR due to its rapid elongation rate， extreme thermostability and outstanding fidelity. Here in this study， we utilized silkworm-baculovirus expression vector system (silkworm-BEVS) to express the recombinant Tkod-Pol (rKOD) with N-terminal (rKOD-N) or C-terminal (rKOD-C) tandem fusion tags. By using BEVS， we produced functional rKODs with satisfactory yields， about 1.1 mg/larva for rKOD-N and 0.25 mg/larva for rKOD-C， respectively. Interestingly， we found that rKOD-C shows higher thermostability at 95 °C than that of rKOD-N， while that rKOD-N is significantly unstable after exposing to long period of heat-shock. We also assessed the polymerase activity as well as the fidelity of purified rKODs under various conditions. Compared with commercially available rKOD， which is expressed in E. coli expression system， rKOD-C exhibited almost the same PCR performance as the commercial rKOD did， while rKOD-N did lower performance. Taken together， our results suggested that silkworm-BEVS can be used to express and purify efficient rKOD in a commercial way.