E. coli Stablelabel S30 lysate for optimized cell-free NMR sample preparation

Cell-free (CF) synthesis with highly productive E. coli lysates is a convenient method to produce labeled proteins for NMR studies. Despite reduced metabolic activity in CF lysates, a certain scrambling of supplied isotope labels is still notable. Most problematic are conversions of N-15 labels of the amino acids L-Asp, L-Asn, L-Gln, L-Glu and L-Ala, resulting in ambiguous NMR signals as well as in label dilution. Specific inhibitor cocktails suppress most undesired conversion reactions, while limited availability and potential side effects on CF system productivity need to be considered. As alternative route to address NMR label conversion in CF systems, we describe the generation of optimized E. coli lysates with reduced amino acid scrambling activity. Our strategy is based on the proteome blueprint of standardized CF S30 lysates of the E. coli strain A19. Identified lysate enzymes with suspected amino acid scrambling activity were eliminated by engineering corresponding single and cumulative chromosomal mutations in A19. CF lysates prepared from the mutants were analyzed for their CF protein synthesis efficiency and for residual scrambling activity. The A19 derivative Stablelabel containing the cumulative mutations asnA, ansA/B, glnA, aspC and ilvE yielded the most useful CF S30 lysates. We demonstrate the optimized NMR spectral complexity of selectively labeled proteins CF synthesized in Stablelabel lysates. By taking advantage of ilvE deletion in Stablelabel, we further exemplify a new strategy for methyl group specific labeling of membrane proteins with the proton pump proteorhodopsin.

Automated summary

Cell-free synthesis using highly productive E. coli lysates is a convenient method for producing labeled proteins for NMR studies. However, there is still a significant scrambling of supplied isotope labels in the lysates. In this study, we describe the generation of optimized E. coli lysates with reduced amino acid scrambling activity to address this issue. We engineered cumulative mutations in specific enzymes suspected of causing amino acid scrambling and demonstrated the usefulness of the resulting lysates in CF protein synthesis.