Cell-free protein expression systems offer many advantages over cell-based
approaches for the expression and isotope labeling of proteins for NMR analysis. Cellfree
systems allow for the rapid single day production of target proteins at milligram
production levels with inexpensive isotope enrichment. The open nature of these
systems also allows the addition of molecules that can aid the folding and stabilization
of target proteins. In this chapter we briefly discuss the available cell-free expression
systems and whether they can be used for the isotope-labeling of a target protein, and
how new PCR-directed cell-free expression approaches can aid the rapid identification
of expression constructs with enhanced yield and solubility. We then focus on recent
advances in the cell–free production of proteins for NMR structural analysis of large
proteins and macromolecular complexes (including membrane proteins). The NMR
spectra of such molecules are often problematic to assign because of their large number
of cross peaks and line broadening resulting in loss in signal resolution and intensity. A
range of selective, combinatorial and segmental isotope labeling strategies based on
cell-free protein synthesis are now available to enable residue-specific and sequencespecific
assignment of NMR spectra. Cell-free deuteration of target proteins can reduce
line broadening issues, and stereo-array isotope labeled (SAIL) amino acids can be
incorporated to provide NOE constraints for structure determination. Cell-free protein
synthesis also allows the incorporation of unnatural amino acids which can act as NMR
probes to provide long distance information.
Keywords: Cell-free protein synthesis, Deuteration, Isotope labeling,
Macromolecular complex, Membrane protein, NMR spectroscopy, SAIL,
Unnatural amino acid.