Nanodiscs are emerging to serve as transfer vectors for the insertion of recombinant membrane proteins into membranes of living cells. In combination with cell-free expression technologies, this novel process opens new perspectives to analyze the effects of even problematic targets such as toxic, hard-to-express, or artificially modified membrane proteins in complex cellular environments of different cell lines. Furthermore, transferred cells must not be genetically engineered and primary cell lines or cancer cells could be implemented as well. We have systematically analyzed the basic parameters of the nanotransfer approach and compared the transfer efficiencies from nanodiscs with that from Salipro particles. The transfer of five membrane proteins was analyzed: the prokaryotic proton pump proteorhodopsin, the human class A family G-protein coupled receptors for endothelin type B, prostacyclin, free fatty acids type 2, and the orphan GPRC5B receptor as a class C family member. The membrane proteins were cell-free synthesized with a detergent-free strategy by their cotranslational insertion into preformed nanoparticles containing defined lipid environments. The purified membrane protein/nanoparticles were then incubated with mammalian cells. We demonstrate that nanodiscs disassemble and only lipids and membrane proteins, not the scaffold protein, are transferred into cell membranes. The process is detectable within minutes, independent of the nanoparticle lipid composition, and the transfer efficiency directly correlates with the membrane protein concentration in the transfer mixture and with the incubation time. Transferred membrane proteins insert in both orientations, N-terminus in and N-terminus out, in the cell membrane, and the ratio can be modulated by engineering. The viability of cells is not notably affected by the transfer procedure, and transferred membrane proteins stay detectable in the cell membrane for up to 3 days. Transferred G-protein coupled receptors retained their functionality in the cell environment as shown by ligand binding, induction of internalization, and specific protein interactions. In comparison to transfection, the cellular membrane protein concentration is better controllable and more uniformly distributed within the analyzed cell population. A further notable difference to transfection is the accumulation of transferred membrane proteins in clusters, presumably determined by microdomain structures in the cell membranes.

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 15N 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.

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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 15N labels of the amino acids L-Asp, LAsn, L-Gln, L-Glu and L-Ala, resulting into ambiguous NMR signals as well as into label dilution. Specific inhibitor cocktails could 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 constructing 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 was finally identified as giving 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 the ilvE deletion in "Stablelabel", we further exemplify a new strategy for methyl group specific labeling of membrane proteins with the proton pump proteorhodopsin.