作者
Catherine M Joyce, ND Grindley
发表日期
1983/4
期刊
Proceedings of the National Academy of Sciences
卷号
80
期号
7
页码范围
1830-1834
简介
Using currently available gene fusion techniques, we have constructed plasmids that direct the overproduction of the carboxyl-terminal two-thirds of DNA polymerase I, corresponding to the proteolytically derived "Klenow fragment." We have obtained overproduction amounting to several percent of the cellular protein using constructs in which expression is directed either from the lac promoter or from the leftward promoter of phage lambda. The polymerase fragment has been purified to homogeneity from such overproducing strains by a rapid three-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. Moreover, the techniques we have described for the cloning …
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CM Joyce, ND Grindley - Proceedings of the National Academy of Sciences, 1983