Advances in cell-free protein synthesis for NMR sample preparation

Abstract

Cell-based expression strategies exist for the2H,13C and15N stable-isotope enrichment of target proteins for NMR studies. However, there are no guarantees that the protein will express to a reasonable level or be folded correctly. In addition, the economic cost of producing an isotope-labeled protein may be prohibitive because of the scale of production required or the cost of isotope-labeling sources. Many of these problems may be alleviated by the use of cell-free protein synthesis systems. The open nature of these systems allows the addition of molecules that can aid the folding and stabilization of the protein being produced. Same-day mg/ml production of isotope-enriched proteins can be achieved cheaply by using isotope-labeled amino acids sparingly in the reaction mix. Cell-free systems based on Escherichia coli and wheat germ extracts have been adapted for the isotope enrichment of proteins. The best expression conditions and construct design for target protein production can be screened on the microlitre scale prior to preparative production on the millilitre scale. Selective and uniform isotope enrichment is achieved by simply replacing the amino acids in the reaction mix with their equivalent labeled versions. Combinatorial selective labeling strategies have been developed to enable residue-specific and sequence-specific assignment of NMR spectra. The study of large proteins and complexes can be facilitated by cell-free deuteration of the target protein(s), or by the stereo-array isotope labeling (SAIL) approach. The open nature of the cell-free system means that subunits of a complex can be exclusively labeled by expressing them in the presence of their unlabeled binding partners. In this way, normally insoluble subunits can be rescued into soluble complexes. A number of strategies also exist for the site-specific incorporation of non-natural amino acids. In the future, these methods will enable the incorporation of novel amino acids that can aid NMR assignment or investigations of functional activity by NMR.