Selective elimination of the exonuclease activity of the deoxyribonucleic acid polymerase from Escherichia coli B by limited proteolysis. Links to PubMed are also available for Selected References. Get a printable copy (PDF file) of the complete article (2.4M), or click on a page image below to browse page by page. Full textįull text is available as a scanned copy of the original print version. Our experience in this study emphasizes the level of ignorance of the factors that influence protein overproduction and the need, in difficult cases, to evaluate many strategies in a semi-empirical manner. Unlike the original construct, the re-engineered derivatives chromatographed as a single species and could be purified to homogeneity in good yield.
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The tendency of the polymerase domain to aggregate was eliminated by re-engineering the protein so as to remove both a solvent-exposed hydrophobic patch and a potentially unstructured region at the extreme N-terminus. By changing the expression system, we were able to obtain the overproduced protein in a soluble form, a necessary first step since attempts to purify the polymerase domain from the insoluble pellet were unsuccessful. These problems were solved by a combination of two distinct strategies. The polymerase domain derivative used in previous studies was insoluble when overproduced and tended to aggregate during purification. coli DNA polymerase I for biochemical and biophysical studies. We describe experiments to produce large quantities of the polymerase domain of E.
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