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 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.
Ozawa
Kiyoshi
K
Research School of Chemistry, Australian National University, Canberra, ACT, 0200, Australia.
Jergic
Slobodan
S
Crowther
Jeffrey A
JA
Thompson
Phillip R
PR
Wijffels
Gene
G
Otting
Gottfried
G
Dixon
Nicholas A
NA
D002474:Cell-Free System; D004258:DNA Polymerase III / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D011485:Protein Binding; D011487:Protein Conformation; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D012680:Sensitivity and Specificity
D029968:Escherichia coli Proteins; D021122:Protein Subunits; D011506:Proteins; D004258:DNA Polymerase III