Cell-free protein synthesis of perdeuterated proteins for NMR studies.
Cell-free protein synthesis protocols for uniformly deuterated proteins typically yield low, non-uniform deuteration levels. This paper introduces an E. coli cell-extract, D-S30, which enables efficient production of proteins with high deuteration levels for all non-labile hydrogen atom positions. Potential applications of the new protocol may include production of proteins with selective isotope-labeling of selected amino acid residues on a perdeuterated background for studies of enzyme active sites or for ligand screening in drug discovery projects, as well as the synthesis of perdeuterated polypeptides for NMR spectroscopy with large supra-molecular structures. As an illustration, it is demonstrated that the 800-kDa chaperonine GroEL synthesized with the D-S30 cell-free system had a uniform deuteration level of about 95% and assembled into its biologically active oligomeric form.
Etezady-Esfarjani
Touraj
T
Institute for Molecular Biology and Biophysics, ETH Zurich, Zurich, Switzerland. touraj@mol.biol.ethz.ch
Hiller
Sebastian
S
Villalba
Cristina
C
Wüthrich
Kurt
K
D018834:Chaperonin 60 / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007553:Isotope Labeling / Q000379:methods; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D014176:Protein Biosynthesis; D022021:Tacrolimus Binding Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification
D018834:Chaperonin 60; D022021:Tacrolimus Binding Proteins