Cotranslational assembly of membrane protein/nanoparticles in cell-free systems

Levin, Roman; Köck, Zoe; Martin, Janosch; Zangl, René; Gewering, Theresa; Schüler, Leah; Moeller, Arne; Dötsch, Volker; Morgner, Nina; Bernhard, Frank

doi:10.1016/j.bbamem.2022.184017

Cited by 7 publications

(5 citation statements)

References 45 publications

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“…A straightforward strategy to prepare such samples could be by cotranslational insertion of CF synthesized membrane proteins directly into membranes of supplied preformed nanodiscs (Levin et al 2020 ; Köck et al 2022 ). However, the systematic evaluation of a suitable lipid composition of the nanodisc membrane, supply of stabilizing ligands into the CF reaction and proper selection of the membrane insertion strategy are essential to obtain high quality samples (Köck et al 2022 ; Levin et al 2022 ).…”

Section: Discussionmentioning

confidence: 99%

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

et al. 2023

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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, 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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Section: Discussionmentioning

confidence: 99%

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

et al. 2023

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“…A straightforward strategy to prepare such samples could be by cotranslational insertion of CF synthesized membrane proteins directly into membranes of supplied preformed nanodiscs 52,53 . However, the systematic evaluation of a suitable lipid composition of the nanodisc membrane, supply of stabilizing ligands into the CF reaction and proper selection of the membrane insertion strategy are essential to obtain high quality samples 53,54 .…”

Section: Discussionmentioning

confidence: 99%

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

Levin

Löhr

Karakoc

et al. 2023

Preprint

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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.

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“…20 These systems offer several advantages, such as enhanced product yield and in high volume, 21 carrying out multi-enzyme reactions in fewer operations, 22 requiring smaller reactor volumes and a wider range of reaction conditions, 23 fast reaction rates and reduced cycle durations, 24 formation of non-natural bioproducts through reconfiguration of the TX-TL systems, 19,25,26 and easy product separation. 27 Several useful commodities such as bio-hydrogen, 26,28 bio-ethanol, 3,10,11,29 protein scaffolds, 30,31 and cell-free proteins including complex proteins, 21,32,33 toxic proteins, 34 membrane proteins, 35–37 functional antibodies, 38 and novel proteins with unnatural amino acids (UNAAs), 39 have been produced through this technology. Furthermore, the development of artificial cell models containing selected characteristics of live cells 40 is a possible gateway towards the development of artificial cells, although still at an early stage of development and currently facing many challenges related to the merging of individual sub-components and the careful tuning of different self-organization and self-assembly processes, owing to the limited information available and the requirement of multidisciplinary expertise.…”

Section: Introductionmentioning

confidence: 99%