PRODUCTION OF A THERMOSTABLE DNA-POLYMERASE BY SITES-SPECIFIC CLEAVAGE OF A HEAT-ELUTED AFFINITY FUSION PROTEIN

A novel strategy is described for bacterial expression and affinity pu rification of a recombinant truncated version of the heat-stable DNA p olymerase I from Thermus aquaticus. The DNA polymerase (Delta Taq) was produced as a fusion to a serum albumin binding affinity handle (ABP) derived from streptococcal protein G. Based on the thermostability of the Delta Taq DNA polymerase, affinity-purified ABP-Delta Taq could b e heat-eluted from HSA columns by incubation at 85 degrees C. To produ ce free Delta Taq DNA polymerase, efficient site-specific cleavage of the affinity tap was performed using a recombinant coxsackievirus 3C p rotease (3C(pro)), also produced as an ABP affinity fusion. Thus, an i ntegrated strategy could be devised where both the cleaved ABP affinit y tag and the protease fusion could be recovered after site-specific c leavage using HSA-affinity chromatography. The flow-through fraction c ontained essentially pure Delta Taq DNA polymerase with full enzymatic activity. (C) 1997 Academic Press.