Site-directed mutagenesis of thelac repressor protein

Paul Edward Wisniowski, Purdue University

Abstract

The lac repressor-lac operator is considered a model for procaryotic gene regulation. The specificity and strength of lac repressor-operator interactions is not completely understood. Due to the inability to crystallize the lac repressor along or with its operator DNA sequence, researchers have turned to other methods of investigation that included genetic studies of mutant lac repressors. These studies have pointed out important domains of the lac repressor including the amino terminal end domain which is responsible for DNA binding. In order to help determine the specific amino acids involved, a mutant lac repressor was produced by site directed mutagenesis. In an attempt to study this mutant lac repressor--a new in vivo laser fluoremetric assay was developed. This laser fluoremetric assay proved to be as sensitive as current pulse labeling experiments. In order to study the lac repressor operator interactions in vitro a modified version of the band shift assay was developed. Combined with a new method of labeling DNA the polymerase chain reaction of the K$\sb{D}$ of the lac repressor was measured at 9.5 $\times$ 10$\sp{-12}$ $\mu$. This is comparable to the filter binding assay which results in a K$\sb{D}$ for the lac repressor 5 $\times$ 10$\sp{-12}$ M. Also, this modified band shift assay was used to study a wider range of salt concentration (0.2 M to 1.0 M KCl) than was possible with the current filter binding assay (0.05 M to 0.25 M).

Degree

Ph.D.

Advisors

Gorenstein, Purdue University.

Subject Area

Biochemistry|Molecular biology

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