Cell-free protein synthesis in an autoinduction system for NMR studies of protein-protein interactions
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 N-15-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 N-15-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 N-15- 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.
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