Cell-free Protein Synthesis in an Autoinduction System for NMR Studies of Protein–Protein Interactions
Source: Journal of Biomolecular NMR, Volume 32, Number 3, July 2005 , pp. 235-241(7)
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 , and subunits of Escherichia coli DNA polymerase III: nascent, selectively 15N-labeled produced in the presence of resulted in a soluble, correctly folded - complex, whereas alone precipitated irrespective of whether was present or not. The 15N-HSQC spectra showed that the N-terminal segment of is mobile in the - complex, yet important for its binding to . 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.
Document Type: Research Article
Affiliations: 1: Research School of Chemistry, Australian National University, Canberra, ACT, 0200, Australia, 2: CSIRO Livestock Industries, Queensland Bioscience Precinct, St. Lucia, Queensland, 4068, Australia, 3: Research School of Chemistry, Australian National University, Canberra, ACT, 0200, Australia, Email: email@example.com
Publication date: July 2005