Cell-Free Protein Synthesis for NMR Structural Analysis of Large Proteins and Complexes

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.