Cell-free expression and selective isotope labelling in protein NMR.
Isotope labelling is a very powerful tool in NMR studies of proteins and has been employed in various ways for over 40 years. 15N and 13C incorporation, using recombinant expression systems, is now commonplace because heteronuclear experiments assist with the fundamental problems of peak resolution and assignment. The use of selective labelling for peak assignment has been restricted by the scrambling of isotope label through metabolic pathways within the expression host organism. The availability of efficient cell-free expression systems with low levels of metabolic conversion allow the increasing use of selective isotope labelling as a tool in protein NMR. We describe two examples, one where a selective labelling scheme can identify backbone amide peaks from unassigned 1H--15N HSQC and HNCO spectra of a 84 residue protein, and another where a specific backbone amide in a 198 residue construct of the ninth and tenth Type III repeats from human fibronectin can be labelled and rapidly identified using a simple HSQC experiment.
Staunton
David
D
Department of Biochemistry, University of Oxford, South Parks Rd, Oxford OX1 3QU, UK. staunton@bioch.ox.ac.uk
Schlinkert
Robin
R
Zanetti
Giulia
G
Colebrook
Simon A
SA
Campbell
Iain D
ID
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D002474:Cell-Free System / Q000737:chemistry / Q000378:metabolism; D005353:Fibronectins / Q000096:biosynthesis / Q000737:chemistry; D006801:Humans; D007553:Isotope Labeling / Q000379:methods*; D019906:Nuclear Magnetic Resonance, Biomolecular; D014176:Protein Biosynthesis; D017433:Protein Structure, Secondary; D011506:Proteins / Q000737:chemistry*
D000596:Amino Acids; D005353:Fibronectins; D011506:Proteins