HEAT-MEDIATED ACTIVATION OF AFFINITY-IMMOBILIZED TAQ DNA-POLYMERASE

A novel strategy for heat-mediated activation of recombinant Tag DNA p olymerase is described A serum albumin binding protein tag is used to affinity-immobilize an E. coli-expressed Tag DNA polymerase fusion pro tein onto a solid support coated with human serum albumin (HSA). Analy sis of heat-mediated elution showed that elevated temperatures (>70 de grees C) were required to significantly release the fusion protein fro m the solid support. A primer-extension assay showed that immobilizati on of the fusion protein resulted in little or no extension product. I n contrast, fusion protein released from the HSA ligand by heat showed high polymerase activity. Thus, a heat-mediated release and reactivat ion of the Tag DNA polymerase fusion protein from the solid support ca n be obtained to allow for hot-start PCR with improved amplification p erformance.