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 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.

Transfer mechanism of cell-free synthesized membrane proteins into mammalian cells.

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.

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

Nanoparticles composed of amphiphilic scaffold proteins and small lipid bilayers are valuable tools for reconstitution and subsequent functional and structural characterization of membrane proteins. In combination with cell-free protein production systems, nanoparticles can be used to cotranslationally and translocon independently insert membrane proteins into tailored lipid environments. This strategy enables rapid generation of protein/nanoparticle complexes by avoiding detergent contact of nascent membrane proteins. Frequently in use are nanoparticles assembled with engineered derivatives of either the membrane scaffold protein (MSP) or the Saposin A (SapA) scaffold. Furthermore, several strategies for the formation of membrane protein/nanoparticle complexes in cell-free reactions exist. However, it is unknown how these strategies affect functional folding, oligomeric assembly and membrane insertion efficiency of cell-free synthesized membrane proteins. We systematically studied membrane protein insertion efficiency and sample quality of cell-free synthesized proteorhodopsin (PR) which was cotranslationally inserted in MSP and SapA based nanoparticles. Three possible PR/nanoparticle formation strategies were analyzed: (i) PR integration into supplied preassembled nanoparticles, (ii) coassembly of nanoparticles from supplied scaffold proteins and lipids upon PR expression, and (iii) coexpression of scaffold proteins together with PR in presence of supplied lipids. Yield, homogeneity as well as the formation of higher PR oligomeric complexes from samples generated by the three strategies were analyzed. Conditions found optimal for PR were applied for the synthesis of a G-protein coupled receptor. The study gives a comprehensive guideline for the rapid synthesis of membrane protein/nanoparticle samples by different processes and identifies key parameters to modulate sample yield and quality.

Insights into population behavior during the COVID-19 pandemic from cell phone mobility data and manifold learning.

Understanding the complex interplay between human behavior, disease transmission and non-pharmaceutical interventions during the COVID-19 pandemic could provide valuable insights with which to focus future public health efforts. Cell phone mobility data offer a modern measurement instrument to investigate human mobility and behavior at an unprecedented scale. We investigate aggregated and anonymized mobility data, which measure how populations at the census-block-group geographic scale stayed at home in California, Georgia, Texas and Washington from the beginning of the pandemic. Using manifold learning techniques, we show that a low-dimensional embedding enables the identification of patterns of mobility behavior that align with stay-at-home orders, correlate with socioeconomic factors, cluster geographically, reveal subpopulations that probably migrated out of urban areas and, importantly, link to COVID-19 case counts. The analysis and approach provide local epidemiologists a framework for interpreting mobility data and behavior to inform policy makers' decision-making aimed at curbing the spread of COVID-19.

Co-translational Insertion of Membrane Proteins into Preformed Nanodiscs.

Cell-free expression systems allow the tailored design of reaction environments to support the functional folding of even complex proteins such as membrane proteins. The experimental procedures for the co-translational insertion and folding of membrane proteins into preformed and defined membranes supplied as nanodiscs are demonstrated. The protocol is completely detergent-free and can generate milligrams of purified samples within one day. The resulting membrane protein/nanodisc samples can be used for a variety of functional studies and structural applications such as crystallization, nuclear magnetic resonance, or electron microscopy. The preparation of basic key components such as cell-free lysates, nanodiscs with designed membranes, critical stock solutions as well as the assembly of two-compartment cell-free expression reactions is described. Since folding requirements of membrane proteins can be highly diverse, a major focus of this protocol is the modulation of parameters and reaction steps important for sample quality such as critical basic reaction compounds, membrane composition of nanodiscs, redox and chaperone environment, or DNA template design. The whole process is demonstrated with the synthesis of proteorhodopsin and a G-protein coupled receptor.

Characterisation of a novel cellobiose 2-epimerase from thermophilic Caldicellulosiruptor obsidiansis for lactulose production.

BACKGROUND: Lactulose, a bioactive lactose derivative, has been widely used in food and pharmaceutical industries. Isomerisation of lactose to lactulose by cellobiose 2-epimerase (CE) has recently attracted increasing attention, since CE produces lactulose with high yield from lactose as a single substrate. In this study, a new lactulose-producing CE from Caldicellulosiruptor obsidiansis was extensively characterised. RESULTS: The recombinant enzyme exhibited maximal activity at pH 7.5 and 70 °C. It displayed high thermostability with Tm of 86.7 °C. The half-life was calculated to be 8.1, 2.8 and 0.6 h at 75, 80, and 85 °C, respectively. When lactose was used as substrate, epilactose was rapidly produced in a short period, and afterwards both epilactose and lactose were steadily isomerised to lactulose, with a final ratio of 35:11:54 for lactose:epilactose:lactulose. When the reverse reaction was investigated using lactulose as substrate, both lactose and epilactose appeared to be steadily produced from the start. CONCLUSION: The recombinant CE showed both epimerisation and isomerisation activities against lactose, making it an alternative promising biocatalyst candidate for lactulose production. © 2016 Society of Chemical Industry.