PURIFICATION OF TRYPTOPHAN REPRESSOR PROTEIN OF ESCHERICHIA COLI: PHYSICAL AND IMMUNOCHEMICAL CHARACTERIZATION
Abstract
Trp repressor protein of Escherichia coli was purified in good yield from cells harboring a plasmid wherein the trpR gene was positioned downstream from a promoter subject to thermal activation. The purification procedure involved polymin P precipitation, gel filtration of a selective salt eluate of the polymin P precipitate, DEAE Sepharose, and hydroxylapatite chromatoraphy. Chemical crosslinking and gel filtration studies indicate that native Trp repressor is a dimer. The protein interacts strongly with the primary trp operator with an equilibrium dissociation constant of 6.7 nM as measured by protein distribution analysis. The repressor also specifically binds to nonpalindromic sequences deleted for either the upstream or downstream half of the primary trp operator in response to L-tryptophan. High titre antiserum directed against Trp repressor protein was produced in rabbits. The antiserum is monospecific towards denatured protein, but crossreacts with one other E. coli protein in nondenatured cell extracts. The binding of L-tryptophan to Trp repressor precipitates a conformational change as demonstrated by: (1) preliminary NMR studies, and (2) altered reactivity of the antiserum towards the repressor-tryptophan complex. In vivo, Trp repressor is a stable protein with a half life of about 36 hours.
Degree
Ph.D.
Subject Area
Biochemistry
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