Cell-free Protein Synthesis in an Autoinduction System for NMR Studies of Protein-protein Interactions

Overview

Journal J Biomol NMR

Publisher Springer

Specialties Molecular Biology
Nuclear Medicine

Date 2005 Sep 1

PMID 16132823

Citations 12

Authors

Kiyoshi Ozawa

Slobodan Jergic

Jeffrey A Crowther

Phillip R Thompson

Gene Wijffels

Gottfried Otting

Nicholas A Dixon

Affiliations

Soon will be listed here.

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 few 15N-HSQC spectra. The concept is demonstrated with binary and ternary complexes between the chi, psi and gamma subunits of Escherichia coli DNA polymerase III: nascent, selectively 15N-labeled psi produced in the presence of chi resulted in a soluble, correctly folded chi-psi complex, whereas psi alone precipitated irrespective of whether gamma was present or not. The 15N-HSQC spectra showed that the N-terminal segment of psi is mobile in the chi-psi complex, yet important for its binding to gamma. 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.

Citing Articles

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Plasmid replication initiator interactions with origin 13-mers and polymerase subunits contribute to strand-specific replisome assembly.

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A direct proofreader-clamp interaction stabilizes the Pol III replicase in the polymerization mode.

Jergic S, Horan N, Elshenawy M, Mason C, Urathamakul T, Ozawa K EMBO J. 2013; 32(9):1322-33.

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A single subunit directs the assembly of the Escherichia coli DNA sliding clamp loader.

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