Construction of a plasmid that overproduces the large proteolytic fragment (Klenow fragment) of DNA polymerase I of Escherichia coli.

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Abstract
Using currently available gene fusion techniques, plasmids were constructed that direct the overproduction of the carboxyl-terminal 2/3 of DNA polymerase I, corresponding to the proteolytically derived Klenow fragment. Overproduction amounting to several percent of the cellular protein was obtained using constructs in which expression is directed from the lac promoter or from the leftward promotor of phage .lambda.. The polymerase fragment was purified to homogeneity from such overproducing strains by a rapid 3-stage purification procedure, yielding material capable of carrying out the same reactions (polymerization, 3'' labeling, DNA sequence analysis) as the proteolytically derived fragment. The availability of such overproducing strains should greatly facilitate structural and mechanistic studies of DNA polymerase I. The techniques described for the cloning and expression of a gene fragment should be generally applicable for the study of protein structure and function in other systems.