REENGINEERING THE POLYMERASE DOMAIN OF KLENOW FRAGMENT AND EVALUATIONOF OVERPRODUCTION AND PURIFICATION STRATEGIES

We describe experiments to produce large quantities of the polymerase domain of E. coli DNA polymerase I for biochemical and biophysical stu dies. The polymerase domain derivative used in previous studies was in soluble when overproduced and tended to aggregate during purification. These problems were solved by a combination of two distinct strategie s. By changing the expression system, we were able to obtain the overp roduced protein in a soluble form, a necessary first step since attemp ts to purify the polymerase domain from the insoluble pellet were unsu ccessful. The tendency of the polymerase domain to aggregate was elimi nated by re-engineering the protein so as to remove both a solvent-exp osed hydrophobic patch and a potentially unstructured region at the ex treme N-terminus. Unlike the original construct, the re-engineered der ivatives chromatographed as a single species and could be purified to homogeneity in good yield. Our experience in this study emphasizes the level of ignorance of the factors that influence protein overproducti on and the need, in difficult cases, to evaluate many strategies in a semi-empirical manner.