Heat-mediated activation of affinity-immobilized Taq DNA polymerase.

A novel strategy for heat-mediated act i vation of recombinant Taq DNA polymeras e is described. A serum albumin binding pro tein tag is used to affinity-immobilize an E . col i-expressed Taq DNA polymerase fusion protein onto a solid support coated with hu man serum albumin (HSA). Analysis o f heat-mediated elution showed that elevated temperatures (>70 ° C) were required to sig nificantly release the fusion protein from th e solid support. A primer-extensi on assa y showed that immobilization of the fusion protein resulted in little or no extension product. In contrast, fusion protein released from the HSA ligand by heat showed high polymerase activity. Thus, a heat-mediated release and reactivation of the Taq DNA polymerase fusion protein from the solid support can be obtained to allow for hot start PCR with improved amplification pe r formance .