Cell-free protein synthesis in an autoinduction system for NMR studies of protein-protein interactions
Date
2005
Authors
Ozawa, Kiyoshi
Jergic, Slobodan
Crowther, Jeffrey
Thompson, Phillip
Wijffels, Gene
Dixon, Nicholas
Otting, Gottfried
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Publisher
Kluwer Academic Publishers
Abstract
Cell-free protein synthesis systems provide facile access to proteins in a nascent state that enables formation of soluble, native protein-protein complexes even if one of the protein components is prone to self-aggregation and precipitation. Combined with selective isotope-labeling, this allows the rapid analysis of protein-protein interactions with few15N-HSQC spectra. The concept is demonstrated with binary and ternary complexes between the χ, ψ and γ subunits of Escherichia coli DNA polymerase III: nascent, selectively15N-labeled ψ produced in the presence of χ resulted in a soluble, correctly folded χ-ψ complex, whereas ψ alone precipitated irrespective of whether γ was present or not. The15N-HSQC spectra showed that the N-terminal segment of ψ is mobile in the χ-ψ complex, yet important for its binding to γ. The sample preparation was greatly enhanced by an autoinduction strategy, where the T7 RNA polymerase needed for transcription of a gene in a T7-promoter vector was produced in situ.
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Keywords
Keywords: DNA directed DNA polymerase alpha; DNA directed DNA polymerase gamma; RNA polymerase; amino terminal sequence; article; bacteriophage T7; cell free system; Escherichia coli; gene vector; genetic transcription; in vitro study; isotope labeling; nitrogen nu 15N-HSQC; Cell-free protein synthesis; DNA polymerase III; Protein folding; Protein-protein interaction
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Source
Journal of Biomolecular NMR
Type
Journal article
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2037-12-31
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