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

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 χ, ψ and γ subunits of Escherichia coli DNA polymerase III: nascent, selectively ¹⁵N-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. The ¹⁵N-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.