M. Y. acknowledges the funding from the Postgraduate Research &#38; Practice Innovation Program of Jiangsu Province, China (Grant No. KYCX22_2803). L.K. is thankful for the support of the Natural Science Research of Jiangsu Higher Education Institutions of China, China (Grant No. 17KJB180003), the Natural Science Foundation of Jiangsu Normal University, China (Grant No. 17XLR037), Priority Academic Program Development of Jiangsu Higher Education Institutions, China, and the Jiangsu Specially-Appointed Professor program, China.

1. Extract preparation
<ol>
	<li>Streak the E. coli BL21 (DE3) strain from a glycerol stock onto a Luria Bertani (LB) agar plate and incubate for at least 15 h at 37 &#176;C.</li>
	<li>Prepare an overnight preculture by inoculating a single colony from the freshly prepared LB plate into a 50 mL flask of Luria Bertani (LB) medium.</li>
	<li>Inoculate 5 mL of preculture into 500 mL of 2xYTPG medium in a 3 L baffled Erlenmeyer flask. Grow it at 37 &#176;C with vigorous shaking (between 220 rpm and 250 rpm) and harvest the cells when they reach the mid-log phase (OD600 ~ 3). Then, place the cell cultures in cold icy water for 10 min and centrifuge at 8,000 &#215; g for 15 min at 4 &#176;C. 
	NOTE: In this step, the glucose in 2xYTPG medium can be omitted for specific applications9,12.</li>
	<li>Resuspend the resulting cell pellets in 35 mL of prechilled S30 buffer A, followed by centrifugation at 8,000 &#215; g for 15 min at 4 &#176;C. Discard the supernatant.</li>
	<li>Repeat step 1.4 twice. 
	NOTE: The cell pellets can be stored at -80 &#176;C if not used immediately.</li>
	<li>Resuspend the final pellets in S30 buffer B (e.g., use 1.1 mL of S30 buffer B per 1 g of cell pellet) and disrupt the cells by one pass through French Press at 17,000 psi.
	<ol>
		<li>For using the French Press, follow the user manual and transfer the cell suspension to the French press metal disruption chamber, ensuring that all components are assembled and the bottom valve of the disruption chamber is closed.</li>
		<li>Transfer the assembled disruption chamber to the hydraulic platform and secure the safety lock.</li>
		<li>Turn on the hydraulic pump and start the disruption.</li>
		<li>Control the outlet valve to enable the cell suspension to flow out of the outlet tube into a new 50 mL tube. Adjust the flow speed to ensure efficient disruption, ideally one drop at a time. Maintain the pressure above the lower limit of 15,000 psi.</li>
	</ol>
	</li>
	<li>Centrifuge the resulting lysate at 30,000 &#215; g for 30 min at 4 &#176;C and collect the supernatant. Repeat the centrifugation once and collect the supernatant. Add 0.3 volume of preincubation buffer into the collected supernatant and incubate with gentle shaking at 37 &#176;C for 80 min. 
	NOTE: The use of preincubation buffer could increase the final yield though it has been reported that an empty run-off (without preincubation buffer) could also improve the efficiency37,38, which depends on the corresponding source strain.</li>
	<li>Dialyze the resulting run off mixture for 2 h against 2 L of S30 buffer C, exchange the dialysis buffer once with 2 L of fresh S30 buffer C, and dialyze overnight at 4 &#176;C39. 
	NOTE: The second step of overnight dialysis can be shortened to approximately 3 h.</li>
	<li>Collect the dialyzed sample and centrifuge at 30,000 &#215; g for 30 min at 4 &#176;C. Collect the supernatant and aliquot into appropriate volumes and flash freeze immediately in liquid nitrogen. 
	NOTE: The frozen sample can be stored at -80 &#176;C at least for 6 months to 1 year without loss of efficiency.</li>
</ol>
2. T7 RNA polymerase
<ol>
	<li>Transform pAR1219 plasmid into BL21(DE3) star E. coli competent cells40.</li>
	<li>Inoculate 10 mL of an overnight culture into 1 L of LB medium (containing 100 &#181;g/mL ampicillin). Grow the cells at 37 &#176;C until OD600 reaches 0.6-0.8.</li>
	<li>Start the induction by adding a final concentration of 1 mM IPTG. Induce the cells for an additional 5 h and harvest by centrifugation at 8,000 &#215; g for 15 min at 4 &#176;C. Store the cell pellets at -80 &#176;C for up to several weeks.</li>
	<li>Resuspend the cell pellets in 30 mL of T7 buffer A and disrupt the cells by one pass through the French Press at 15,000 psi. Remove the cell debris by centrifugation at 20,000 &#215; g for 30 min at 4 &#176;C.</li>
	<li>Add streptomycin sulfate drop by drop to the supernatant from the previous step, reaching a final concentration of 4% (w/v). After a short incubation on ice, centrifuge at 20,000 &#215; g for 30 min at 4 &#176;C.</li>
	<li>Filter the supernatant through a 0.45 &#181;m filter membrane and load the filtered sample onto a strong anion exchange column (column volume of 40 mL), which was preequilibrated with 10 column volumes of T7 buffer B, via an automated liquid chromatograph system.</li>
	<li>Wash the loaded column with 50 column volumes of T7 buffer B and elute the sample with 10 column volumes of low salt (50 mM NaCl) and high salt (500 mM) T7 buffer C mixtures, establishing a linear concentration gradient of NaCl from 50 to 500 mM at a flow rate of ~3 mL/min41.</li>
	<li>Collect the peak fractions and analyze by SDS-PAGE. 
	NOTE: T7 polymerase exhibits a predominate band at ~100 kDa, while significant amounts of impurities still appear on the SDS-PAGE gel.</li>
	<li>Pool the fractions containing T7 polymerase and dialyze against 2 L of T7 buffer C overnight.</li>
	<li>Add glycerol to reach a final concentration of 10% and concentrate the resulting mixture to a final concentration of 3-4 mg/mL by ultrafiltration42. 
	NOTE: If precipitation appears during the concentration process, stop immediately and remove the precipitates by centrifugation.</li>
	<li>Adjust the glycerol concentration to 50%. Aliquot and flash freeze the sample with liquid nitrogen. Store at &#8722;80 &#176;C for a longer period. 
	NOTE: A small aliquot could also be stored at -20 &#176;C for at least 1 month without loss of efficiency.</li>
</ol>
3. Buffer preparation
<ol>
	<li>Prepare buffers as indicated in Table 1 a day before usage.</li>
</ol>
4. Template design and preparation
<ol>
	<li>Clone the gene of interest into a T7 promoter-based vector or generate a linear PCR product containing the gene of interest. 
	NOTE: See the discussion section for design principles.</li>
	<li>Prepare the plasmid from an overnight culture using a plasmid extraction kit. 
	NOTE: We recommend selecting a plasmid kit that includes an isopropanol precipitation step followed by a washing procedure with 70% ethanol.</li>
	<li>Dissolve the dried DNA in a small volume of ultrapure water to a concentration between 200 &#181;g/mL and 500 &#181;g/mL, as determined by a micro volume spectrophotometer.</li>
	<li>Optional: Directly express proteins from linear PCR products. 
	NOTE: In this case, a PCR purification step is needed.</li>
</ol>
5. Mg2+ and K+ optimization
<ol>
	<li>Prepare a master mix for Mg2+ concentration screening as indicated in Table 2, or for K+ concentration screening as indicated in Table 3.</li>
	<li>Transfer the master mix into individual microfuge tubes or a V-shaped 96-well plate.</li>
	<li>Pipette the exact volume of Mg2+ or K+ stock solutions into individual microfuge tubes or the V-shaped 96-well plate and complete the CFPS reactions with ultrapure water. Incubate the reactions for at least 2 h. 
	NOTE: If using a V-shaped 96-well plate, seal the plate with a plastic cover to prevent evaporation.</li>
	<li>Take out 2 &#181;L of the reaction mixture and transfer into a black 96-well plate for fluorescence measurements by a plate reader. 
	NOTE: We use a fluorescent plate reader with the following setup: excitation/emission: 485/528, Gain: 50, Read height: 7.00 mm. However, one would need to tune their plate reader to generate a specific calibration curve using purified fluorescent proteins.</li>
</ol>
6. Encapsulation
<ol>
	<li>Droplet
	<ol>
		<li>Prepare a surfactant-containing fluorinated oil (2% PFPE-PEG in HFE7500) or a lipid-mineral oil solution by dissolving lipid in mineral oil (refer to step 6.2.1)</li>
		<li>Prepare a total volume of 100 &#181;L of CFPS reaction by combining the corresponding reagents 2-18 from Table 4. Add CFPS reaction to 500 &#181;L of the previously prepared oil in a 1.5 mL tube and then, rub the tube vigorously on the tube rack 50x to form fine (water-in-oil) droplets. Incubate the tube at 30 &#176;C to perform the reaction. 
		NOTE: Simple microfluidics chips could also be used to generate homogenous droplets as well43.</li>
	</ol>
	</li>
	<li>Giant unilamellar vesicles (GUVs)
	<ol>
		<li>Preparing the lipid-mineral oil solution
		<ol>
			<li>Add 57 &#181;L of chloroform into a 4 mL glass vial and then add 18 &#181;L of 25 mg/mL 1-palmitoyl-2-oleoyl-glycerol-3-phosphocholine (POPC) lipids to form the chloroform lipid solution, achieving a final concentration of 8 mM (use other lipids with similar final concentrations). 
			NOTE: This mixing step was to ensure homogeneous mixing of different lipids.</li>
			<li>Evaporate the chloroform under an argon flow for 15 min, and then, further evaporate under vacuum for 1 h.</li>
			<li>Dissolve the resulting dry lipids in 1,500 &#181;L of mineral oil, reaching a final concentration of 400 &#181;M. Incubate the mixture overnight at room temperature.</li>
		</ol>
		</li>
		<li>Forming an interfacial lipid layer
		<ol>
			<li>Add 500 &#181;L of the outer solution (see Table 5) to a 1.5 mL tube, and slowly layer 250 &#181;L of lipid-mineral oil solution on top of the outer solution. Incubate at room temperature for 30 min to form a stable interfacial lipid layer.</li>
		</ol>
		</li>
		<li>Preparation of the inner solution
		<ol>
			<li>Mix all the reagents in Table 6 to form the preinner solution and keep it on ice until use. Complete the inner solution by adding T7RNAP, S30 extract, and DNA template into the preinner solution as reagents 16-18 listed in Table 4. 
			NOTE: Increasing encapsulated sucrose concentration above 250 mM might inhibit cell-free translation44.</li>
		</ol>
		</li>
		<li>Formation of GUVs
		<ol>
			<li>Add 50 &#181;L of inner solution to a 1.5 mL new tube containing 500 &#181;L of lipid-mineral oil, pipette up and down rapidly, and vortex vigorously.</li>
			<li>Leave the tube on ice for 10 min and slowly add 20 &#181;L of the emulsion mixture to the top of the oil phase in the 1.5 mL tube from step 6.2.2.</li>
			<li>Centrifuge at 800 &#215; g (use a centrifugation speed ranging from 100 &#215; g to 1000 &#215; g) for 10 min at 4 &#176;C and observe the formation of GUVs at the bottom of the tube. 
			NOTE: A higher specific centrifugation speed could be used; however, one would consider that the centrifugation speed could influence the size of formed GUVs45.</li>
			<li>Remove the upper oil phase.</li>
			<li>Aspirate 30 &#181;L of GUVs at the bottom of the tube carefully. Incubate the collected GUVs at 30 &#176;C. 
			NOTE: The optimal incubation temperature varies for different proteins.</li>
			<li>Use Confocal Laser Scanning Microscopy (LSM) to monitor the expression of fluorescent proteins.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Supported lipid bilayer (SLB)
	<ol>
		<li>Preparation of SLB chambers
		<ol>
			<li>Piranha-clean 24 x 24 mm #1.5 coverslips by adding seven drops of sulfuric acid and two drops of 50% hydrogen peroxide to the center of each cover slide. Incubate the reactants on the coverslips for at least 45 min; rinse thoroughly with ultrapure water.</li>
			<li>Assemble the reconstitution chamber by attaching a cut 0.5 mL microfuge tube onto the cleaned coverslips using optical glue cured under an ultraviolet lamp (365 nm) for 10 min to form a reaction chamber.</li>
		</ol>
		</li>
		<li>SLB formation
		<ol>
			<li>Dissolve 80 mol% 1,2-dioleoyl-sn-glycerol-3-phosphocholine (DOPC), 19.95 mol% 1,2-dioleoyl-sn-glycerol-3-phospho-L-serine (DOPS), and 0.05 mol% Atto488-DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine = DOPE), mix in chloroform, dry under mild nitrogen flow and vacuum for 1 h to remove the solvent completely.</li>
			<li>Rehydrate the dried lipid film in SLB buffer A, resulting in a final lipid concentration of 4 mg/mL.</li>
			<li>Vortex and sonicate the resulting samples until they become clear.</li>
			<li>Dilute 4 mg/mL of SUVs with 130 &#181;L of SLB buffer A, transfer 75 &#181;L of the suspension to the preformed reaction chambers, and incubate it on a heat block at 37 &#176;C for 1 min. Add 150 &#181;L of SLB buffer A to the reaction chamber for further incubation for 2 min.</li>
			<li>Wash the chamber with 2 mL of SLB buffer B (SLB buffer A without MgCl2) prior to a buffer exchange to the S30 buffer C with 0.4% (w/v) BSA for CFPS reactions, leaving 100 &#181;L of buffer inside the chamber to prevent the formed SLB from drying out.</li>
			<li>Remove the residual S30 buffer C and add the CFPS reaction mixture into the SLB chamber carefully. Incubate the chamber at 30 &#176;C and monitor the expression under a confocal laser scanning microscope.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

CFPS Protocols for Micro-Compartmentalized Synthetic Cells

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

This manuscript outlines a modified Cell-Free Protein Synthesis (CFPS) system designed for use in various micro-compartments across synthetic cell platforms, including water-in-oil droplets, GUVs, and SLBs. We utilized the standard E. coli recombinant protein expression host strain, BL21(DE3), as the source extract for constructing protein-centric synthetic cell systems. This approach yielded approximately 0.5 mg/mL of protein across different compartments. While other customized extract source strains could be ...

The synthesis of synthetic or artificial cells has emerged as a highly prominent field of interdisciplinary research, attracting substantial interest from scientists across the domains of synthetic biology, chemistry, and biophysics. These scientists are united by the common goal of constructing a minimal living cell1,2,3. The rapid growth of this field has been in step with significant advancements in critical technologies, such as recombinant DNA manipulation4, biomimetic materials5, and microfabrication techniques for compartmentalization6, including the Cell-Free Protein Synthesis (CFPS) method7. CFPS systems encompass the essential cellular machinery for transcription and translation, providing the foundational framework for the development and integration of multifunctional artificial cells.
Although CFPS techniques are frequently used in the assembly of synthetic cells, developing a robust and tailored CFPS system for the assembly of various synthetic cell systems remains a complex challenge. Currently, numerous CFPS systems are available, derived from both prokaryotes and eukaryotes model organisms8, each specialized for particular applications in synthetic cell synthesis. Beyond their central roles in transcription and translation, CFPS systems vary in their main components and associated preparation procedures. These variations, which include differences in cell extracts, RNA polymerases, template preparation methods, and buffer compositions, are largely due to the distinct development trajectories pursued by research groups that have intensively optimized their systems for maximal protein yield.
Among the various components of the CFPS system, the cell extract is a critical enzymatic pool for transcription and translation, and thus a key determinant of CFPS performance9. Escherichia coli (E. coli)-based CFPS is the most commonly utilized system due to its status as the best-understood prokaryotic organism. Furthermore, a fully reconstituted CFPS system comprising individually purified proteins and ribosomes, known as PURE10, has been developed by Ueda&#39;s research group, which is particularly suited for applications requiring a clear background. Today, even E. coli-based CFPS systems have diversified, especially in terms of the source strains for the extrac11 and methods of preparation12,13, RNA polymerase14,15, energy sources16,17, and buffer systems18,19. The most frequently used strains include K12 and B strain derivatives, such as A1920, JM10921, BL21 (DE3)22, and Rossetta223, alongside their genetically modified counterparts.
Initially, E. coli strains with reduced RNase and protease activities were chosen to enhance mRNA stability and the stability of newly synthesized recombinant proteins, leading to increased final protein yields24. Subsequently, E. coli extracts were engineered to facilitate specific post-translational modifications, including glycosylation25, phosphorylation26, and lipidation27, were developed to achieve the above posttranslational modifications. Additionally, an array of additives such as molecular chaperons28 and chemical stabilizers have been incorporated to aid the folding of target proteins, contributing to the diversification of CFPS systems. The bacteriophage T7 RNA polymerase, known for its high processivity, is predominantly employed for transcription, although other polymerases such as SP629 have also been utilized. E. coli endogenous RNA polymerase has been adapted for the prototyping of genetic circuits leveraging sigma factors30. Lastly, a variety of energy precursors31,32,33 and different salts and buffer components19,34,35 have been systematically optimized to enhance productivity.
Besides the CFPS system itself, the encapsulation methods as well as compartmentalization materials are also vital for the successful synthetic cell assembly. Various systems that have been developed to successfully encapsulate the CFPS reaction include surfactant-stabilized water/oil droplets, lipid/polymer, and their hybrid unilamellar vesicles (with diameters ranging from 50 nm to several &#956;m), as well as planar-supported lipid bilayers. However, due to the complexed molecule content of the CFPS system, the success rate of encapsulation depends on specific cases, particularly for the formation of vesicles. To improve the success rate and efficiency of encapsulation of CFPS, various microfluid chips have been developed to facilitate the formation of both droplets and vesicles36. Nevertheless, additional chips and devices will need to be established.
This protocol delineates an E. coli CFPS system utilizing the BL21(DE3) strain, which is a commonly employed host for recombinant protein production. The protocol encompasses a detailed account of the cell extract preparation, template preparation, and standard expression optimization using a fluorescent reporter protein. Moreover, we present exemplary outcomes achieved by encapsulating the CFPS system within diverse micro-compartments, including monolayer droplets, double emulsion vesicles, and chambers situated atop supported lipid bilayers. Finally, we expound upon the pivotal procedural elements and the requisite conditions indispensable for the successful establishment of these CFPS systems within distinct environmental contexts.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1,2-dioleoyl-sn-glycero-3-phosphocholine(DOPC)</td>
			<td>Avanti</td>
			<td>850375P</td>
			<td></td>
		</tr>
		<tr>
			<td>1,2-dioleoyl-sn-glycero-3-phospho-L-serine (sodium salt)(DOPS)</td>
			<td>Avanti</td>
			<td>840035P</td>
			<td></td>
		</tr>
		<tr>
			<td>1,4 dithiothreitol (DTT)</td>
			<td>Sigma-Aldrich</td>
			<td>1.11474</td>
			<td></td>
		</tr>
		<tr>
			<td>1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)</td>
			<td>Avanti</td>
			<td>850457P</td>
			<td></td>
		</tr>
		<tr>
			<td>3,5-cyclic AMP (cAMP)</td>
			<td>Sigma-Aldrich</td>
			<td>A9501</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL tubes</td>
			<td>Eppendorf</td>
			<td>Eppendorf Tubes BioBased</td>
			<td></td>
		</tr>
		<tr>
			<td>50% hydrogen peroxide</td>
			<td>Sigma-Aldrich</td>
			<td>516813</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetate</td>
			<td>Sigma-Aldrich</td>
			<td>A6283</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar powder</td>
			<td>Sigma-Aldrich</td>
			<td>05040</td>
			<td></td>
		</tr>
		<tr>
			<td>Alanin</td>
			<td>Sigma-Aldrich</td>
			<td>A4349</td>
			<td></td>
		</tr>
		<tr>
			<td>Amicon Stirred Cells</td>
			<td>MerckMillipore</td>
			<td>UFSC05001</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium acetate (NH4OAc)</td>
			<td>Sigma-Aldrich</td>
			<td>A7262</td>
			<td></td>
		</tr>
		<tr>
			<td>Arginin</td>
			<td>Sigma-Aldrich</td>
			<td>A4474</td>
			<td></td>
		</tr>
		<tr>
			<td>Asparagin</td>
			<td>Sigma-Aldrich</td>
			<td>A0884</td>
			<td></td>
		</tr>
		<tr>
			<td>Aspartat</td>
			<td>Sigma-Aldrich</td>
			<td>A5474</td>
			<td></td>
		</tr>
		<tr>
			<td>ATP</td>
			<td>Roche</td>
			<td>11140965001</td>
			<td></td>
		</tr>
		<tr>
			<td>Atto 488 DOPE</td>
			<td>Sigma-Aldrich</td>
			<td>67335</td>
			<td></td>
		</tr>
		<tr>
			<td>Atto 647N DOPE</td>
			<td>Sigma-Aldrich</td>
			<td>42247</td>
			<td></td>
		</tr>
		<tr>
			<td>Baffled Erlenmeyer flask</td>
			<td>Shuniu</td>
			<td>250 mL, 1000mL</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin(BSA)</td>
			<td>Roche</td>
			<td>10711454001</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifugetube</td>
			<td>Eppendorf</td>
			<td>Eppendorf Tubes 3810X</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifugetube rack</td>
			<td>Eppendorf</td>
			<td>0030119819</td>
			<td></td>
		</tr>
		<tr>
			<td>Chemiluminescence and epifluorescence imaging system</td>
			<td>Uvitec</td>
			<td>Alliance Q9 Advanced</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Sigma-Aldrich</td>
			<td>288306</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal Laser Scanning Microscopy (LSM)</td>
			<td>ZEISS</td>
			<td>LSM 780</td>
			<td></td>
		</tr>
		<tr>
			<td>Countess Cell Counting Chamber Slides</td>
			<td>Thermo Fisher Scientific</td>
			<td>C10283</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslip</td>
			<td>Thermo Scientific</td>
			<td>Menzel BB02400500A113MNZ0</td>
			<td></td>
		</tr>
		<tr>
			<td>creatine kinase (CK)</td>
			<td>Roche</td>
			<td>10127566001</td>
			<td></td>
		</tr>
		<tr>
			<td>Creatine phosphate (CP)</td>
			<td>Sigma-Aldrich</td>
			<td>10621714001</td>
			<td></td>
		</tr>
		<tr>
			<td>Culture dish</td>
			<td>Huanqiu</td>
			<td>90 mm</td>
			<td></td>
		</tr>
		<tr>
			<td>Cystein</td>
			<td>Sigma-Aldrich</td>
			<td>C5360</td>
			<td></td>
		</tr>
		<tr>
			<td>Cytidine 5&#39;-triphosphate disodium salt (CTP)</td>
			<td>aladdin</td>
			<td>C101487</td>
			<td></td>
		</tr>
		<tr>
			<td>Dialysis membrane</td>
			<td>Spectrum</td>
			<td>Standard RC Tubing MWCO: 12-14 kD</td>
			<td></td>
		</tr>
		<tr>
			<td>E.Z.N.A. Cycle Pure Kit</td>
			<td>Omega Bio-Tek</td>
			<td>D6492-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Electro-Heating Standing-Temperature Cultivator</td>
			<td>Yiheng instrument</td>
			<td>DHP-9602</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid(EDTA)</td>
			<td>Biosharp</td>
			<td>1100027</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescent plate reader</td>
			<td>BioTek</td>
			<td>Synergy 2</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorinated oil</td>
			<td>Suzhou CChip scientific instrument</td>
			<td>2%HFE7500</td>
			<td></td>
		</tr>
		<tr>
			<td>Folinic acid</td>
			<td>Sigma-Aldrich</td>
			<td>47612</td>
			<td></td>
		</tr>
		<tr>
			<td>French Press</td>
			<td>G.Heinemann</td>
			<td>HTU-DIGI-Press</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G7021</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamat</td>
			<td>Sigma-Aldrich</td>
			<td>G5667</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamin</td>
			<td>Sigma-Aldrich</td>
			<td>G5792</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma-Aldrich</td>
			<td>G5516</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycin</td>
			<td>Sigma-Aldrich</td>
			<td>G7126</td>
			<td></td>
		</tr>
		<tr>
			<td>Guanosine 5&#39;-triphosphate sodium salt hydrate(GTP)</td>
			<td>Roche</td>
			<td>10106399001</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr>
			<td>HiPrep Q FF 16/10</td>
			<td>Cytiva</td>
			<td>28936543</td>
			<td></td>
		</tr>
		<tr>
			<td>Histidin</td>
			<td>Sigma-Aldrich</td>
			<td>H6034</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoleucin</td>
			<td>Sigma-Aldrich</td>
			<td>I5281</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropyl-&#946;-D-thiogalactopyranoside (IPTG)</td>
			<td>Sigma-Aldrich</td>
			<td>I5502</td>
			<td></td>
		</tr>
		<tr>
			<td>K2HPO4</td>
			<td>Sigma-Aldrich</td>
			<td>P8281</td>
			<td></td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Sigma-Aldrich</td>
			<td>P5655</td>
			<td></td>
		</tr>
		<tr>
			<td>Leucin</td>
			<td>Sigma-Aldrich</td>
			<td>L6914</td>
			<td></td>
		</tr>
		<tr>
			<td>Lysin</td>
			<td>Sigma-Aldrich</td>
			<td>L5501</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium acetate tetrahydrate (Mg(OAc)2 )</td>
			<td>Sigma-Aldrich</td>
			<td>M5661</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride(MgCl2)</td>
			<td>Sigma-Aldrich</td>
			<td>M2670</td>
			<td></td>
		</tr>
		<tr>
			<td>Methionin</td>
			<td>Sigma-Aldrich</td>
			<td>M8439</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge</td>
			<td>Eppendorf</td>
			<td>5424 R</td>
			<td></td>
		</tr>
		<tr>
			<td>Mineral oil</td>
			<td>Sigma-Aldrich</td>
			<td>M5904</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini-PROTEAN Tetra Cell Systems</td>
			<td>Bio-Rad</td>
			<td>1645050</td>
			<td></td>
		</tr>
		<tr>
			<td>Multipurpose Centrifuge</td>
			<td>Eppendorf</td>
			<td>5810 R</td>
			<td></td>
		</tr>
		<tr>
			<td>NaN3</td>
			<td>Sigma-Aldrich</td>
			<td>S2002</td>
			<td></td>
		</tr>
		<tr>
			<td>Nucleic Acid &#38; Protein UV-Assay Measurements</td>
			<td>IMPLEN</td>
			<td>NanoPhotometer N60</td>
			<td></td>
		</tr>
		<tr>
			<td>NucleoBond Xtra Maxi kit for transfection-grade plasmid DNA</td>
			<td>MACHEREY-NAGEL</td>
			<td>740414.5</td>
			<td></td>
		</tr>
		<tr>
			<td>Nunc-Immuno MicroWell 96 well polystyrene plates</td>
			<td>Sigma-Aldrich</td>
			<td>P8616</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR Thermal Cycler</td>
			<td>Eppendorf</td>
			<td>Mastercycler nexus</td>
			<td></td>
		</tr>
		<tr>
			<td>Peptone</td>
			<td>Sigma-Aldrich</td>
			<td>83059</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenylalanin</td>
			<td>Sigma-Aldrich</td>
			<td>P8740</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphoenolpyruvat (PEP)</td>
			<td>GLPBIO</td>
			<td>GC44635</td>
			<td></td>
		</tr>
		<tr>
			<td>PMSF</td>
			<td>Sigma-Aldrich</td>
			<td>PMSF-RO</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyethylene glycol 8000 (PEG 8000)</td>
			<td>Sigma-Aldrich</td>
			<td>89510</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Acetate(KOAc)</td>
			<td>Sigma-Aldrich</td>
			<td>P5708</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride(KCl)</td>
			<td>Sigma-Aldrich</td>
			<td>P9541</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium glutamate (K-glutamate)</td>
			<td>Sigma-Aldrich</td>
			<td>G1501</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium hydroxide(KOH)</td>
			<td>Sigma-Aldrich</td>
			<td>221473</td>
			<td></td>
		</tr>
		<tr>
			<td>Prolin</td>
			<td>Sigma-Aldrich</td>
			<td>P8865</td>
			<td></td>
		</tr>
		<tr>
			<td>Pyruvate kinase (PK)</td>
			<td>Sigma-Aldrich</td>
			<td>P9136</td>
			<td></td>
		</tr>
		<tr>
			<td>Serin</td>
			<td>Sigma-Aldrich</td>
			<td>S4311</td>
			<td></td>
		</tr>
		<tr>
			<td>Shaker</td>
			<td>Zhichushakers</td>
			<td>ZQZY-AF8</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride(NaCl)</td>
			<td>Sigma-Aldrich</td>
			<td>S5886</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide(NaOH)</td>
			<td>Sigma-Aldrich</td>
			<td>S5881</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>aladdin</td>
			<td>S112226</td>
			<td></td>
		</tr>
		<tr>
			<td>Sulfuric acid</td>
			<td>Sigma-Aldrich</td>
			<td>339741</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe Filters</td>
			<td>Jinteng</td>
			<td>0.45 &#956;m</td>
			<td></td>
		</tr>
		<tr>
			<td>Test tube</td>
			<td>Shuniu</td>
			<td>20 mL</td>
			<td></td>
		</tr>
		<tr>
			<td>TGX FastCast Acrylamide Kit, 12%</td>
			<td>Bio-Rad</td>
			<td>#1610175</td>
			<td></td>
		</tr>
		<tr>
			<td>ThermoMixer</td>
			<td>Eppendorf</td>
			<td>ThermoMixer C</td>
			<td></td>
		</tr>
		<tr>
			<td>Threonin</td>
			<td>Sigma-Aldrich</td>
			<td>T8441</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris base</td>
			<td>Sigma-Aldrich</td>
			<td>V900483</td>
			<td></td>
		</tr>
		<tr>
			<td>tRNA</td>
			<td>Roche</td>
			<td>10109550001</td>
			<td></td>
		</tr>
		<tr>
			<td>Tryptone</td>
			<td>Sigma-Aldrich</td>
			<td>T7293</td>
			<td></td>
		</tr>
		<tr>
			<td>Tryptophan</td>
			<td>Sigma-Aldrich</td>
			<td>T8941</td>
			<td></td>
		</tr>
		<tr>
			<td>Tyrosin</td>
			<td>Sigma-Aldrich</td>
			<td>T8566</td>
			<td></td>
		</tr>
		<tr>
			<td>UTP Trisodium salt (UTP)</td>
			<td>aladdin</td>
			<td>U100365</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum Pump with Circulated Water System</td>
			<td>Zhengzhou Greatwall Scientific Industrial and Trade Co.Ltd</td>
			<td>SHB-<img alt="Equation 1" src="/files/ftp_upload/66626/66626eq01.jpg" style="margin: auto;" /></td>
			<td></td>
		</tr>
		<tr>
			<td>Valin</td>
			<td>Sigma-Aldrich</td>
			<td>V4638</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex Mixers</td>
			<td>Kylin-Bell</td>
			<td>Vortex QL-861</td>
			<td></td>
		</tr>
		<tr>
			<td>Water purification system</td>
			<td>MerckMillipore</td>
			<td>Direct ultrapure water (Type 1)</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast extract</td>
			<td>Sigma-Aldrich</td>
			<td>70161</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;-mercaptoethanol</td>
			<td>Sigma-Aldrich</td>
			<td>444203</td>
			<td></td>
		</tr>
	</tbody>
</table>

cell-free protein synthesis system for building synthetic cells

The Cell-Free Protein Synthesis (CFPS) system has been widely employed to facilitate the bottom-up assembly of synthetic cells. It serves as the host for the core machinery of the Central Dogma, standing as an optimal chassis for the integration and assembly of diverse artificial cellular mimicry systems. Despite its frequent use in the fabrication of synthetic cells, establishing a tailored and robust CFPS system for a specific application remains a nontrivial challenge. In this methods paper, we present a comprehensive protocol for the CFPS system, routinely employed in constructing synthetic cells. This protocol encompasses key stages in the preparation of the CFPS system, including the cell extract, template preparation, and routine expression optimization utilizing a fluorescent reporter protein. Additionally, we show representative results by encapsulating the CFPS system within various micro-compartments, such as monolayer droplets, double-emulsion vesicles, and chambers situated atop supported lipid bilayers. Finally, we elucidate the critical steps and conditions necessary for the successful assembly of these CFPS systems in distinct environments. We expect that our approach will facilitate the establishment of good working practices among various laboratories within the continuously expanding synthetic cell community, thereby accelerating progress in the field of synthetic cell development.

For each new batch of cell extract and T7 RNA polymerase, it is recommended to perform a basic screening of both Mg2+ and K+ concentrations to ensure the optimal performance of the CFPS system. The fluorescence of superfolder GFP can serve as an indicator of the overall yield of the CFPS system under varying conditions, as illustrated in Figure 1A,B. Additionally, a parallel yield comparison of the CFPS system across different compartments is shown in <...

Read this Scientific Article Protocol about Cell-free Protein Synthesis System for Building Synthetic Cells at JoVE.com

Author Spotlight: Optimizing CFPS Systems for Synthetic Cell Construction

This protocol describes the Cell-Free Protein Synthesis (CFPS) system used in constructing synthetic cells. It outlines key stages with representative results in different micro-compartments. The protocol aims to establish best practices for diverse laboratories in the synthetic cell community, advancing progress in synthetic cell development.

Cell-Free Protein Synthesis System for Building Synthetic Cells

The Cell-Free Protein Synthesis (CFPS) system has been widely employed to facilitate the bottom-up assembly of synthetic cells. It serves as the host for ...

cell-free-protein-synthesis-system-for-building-synthetic-cells

Research

JoVE Journal

Biochemistry

1.7K Views.  Jiangsu Normal University. This protocol describes the Cell-Free Protein Synthesis (CFPS) system used in constructing synthetic cells. It outlines key stages with representative results in different micro-compartments. The protocol aims to establish best practices for diverse laboratories in the synthetic cell community, advancing progress in synthetic cell development.

Video: Author Spotlight: Optimizing CFPS Systems for Synthetic Cell Construction

Protein Complex Affinity Capture from Cryomilled Mammalian Cells

Affinity capture is an effective technique for isolating endogenous protein complexes for further study. When used in conjunction with an antibody, this technique is also frequently referred to as immunoprecipitation. Affinity capture can be applied in a bench-scale and in a high-throughput context. When coupled with protein mass spectrometry, affinity capture has proven to be a workhorse of interactome analysis. Although there are potentially many ways to execute the numerous steps involved, the following protocols implement our favored methods. Two features are distinctive: the use of cryomilled cell powder to produce cell extracts, and antibody-coupled paramagnetic beads as the affinity medium. In many cases, we have obtained superior results to those obtained with more conventional affinity capture practices. Cryomilling avoids numerous problems associated with other forms of cell breakage. It provides efficient breakage of the material, while avoiding denaturation issues associated with heating or foaming. It retains the native protein concentration up to the point of extraction, mitigating macromolecular dissociation. It reduces the time extracted proteins spend in solution, limiting deleterious enzymatic activities, and it may reduce the non-specific adsorption of proteins by the affinity medium. Micron-scale magnetic affinity media have become more commonplace over the last several years, increasingly replacing the traditional agarose- and Sepharose-based media. Primary benefits of magnetic media include typically lower non-specific protein adsorption; no size exclusion limit because protein complex binding occurs on the bead surface rather than within pores; and ease of manipulation and handling using magnets.

Here we describe protocols to disrupt mammalian cells by solid-state milling at a cryogenic temperature, produce a cell extract from the resulting cell powder, and isolate protein complexes of interest by affinity capture upon antibody-coupled micron-scale paramagnetic beads.

Affinity capture is an effective technique for isolating endogenous protein complexes for further study. When used in conjunction with an antibody, this ...

A Modified Yeast-one Hybrid System for Heteromeric Protein Complex-DNA Interaction Studies

Over the years, the yeast one-hybrid assay has proven to be an important technique for the identification and validation of physical interactions between proteins such as transcription factors (TFs) and their DNA target. The method presented here utilizes the underlying concept of the Y1H but is modified further to study and validate protein complexes binding to their target DNA. Hence, it is referred to as the modified yeast one-hybrid (Y1.5H) assay. This assay is cost effective and can be easily performed in a regular laboratory setting. Albeit using a heterologous system, the described method could be a valuable tool to test and validate the heteromeric protein complex binding to their DNA target(s) for functional genomics in any system of study, especially plant genomics.

The modified yeast one-hybrid assay described here is an extension of the classical yeast one-hybrid (Y1H) assay to study and validate the heteromeric protein complex-DNA interaction in a heterologous system for any functional genomics study.

Over the years, the yeast one-hybrid assay has proven to be an important technique for the identification and validation of physical interactions between ...

Dissipative Microgravimetry to Study the Binding Dynamics of the Phospholipid Binding Protein Annexin A2 to Solid-supported Lipid Bilayers Using a Quartz Resonator

The dissipative quartz crystal microbalance technique is a simple and label-free approach to measure simultaneously the mass uptake and viscoelastic properties of the absorbed/immobilized mass on sensor surfaces, allowing the measurements of the interaction of proteins with solid-supported surfaces, such as lipid bilayers, in real-time and with a high sensitivity. Annexins are a highly conserved group of phospholipid-binding proteins that interact reversibly with the negatively charged headgroups via the coordination of calcium ions. Here, we describe a protocol that was employed to quantitatively analyze the binding of annexin A2 (AnxA2) to planar lipid bilayers prepared on the surface of a quartz sensor. This protocol is optimized to obtain robust and reproducible data and includes a detailed step-by-step description. The method can be applied to other membrane-binding proteins and bilayer compositions.

Here, we present an experimental protocol that can be employed to determine the binding affinities and mode of interaction of label-free phospholipid-binding protein annexin A2 with immobilized solid-supported bilayers (SLB) by simultaneously measuring the mass uptake and the viscoelastic properties of the protein annexin A2.

The dissipative quartz crystal microbalance technique is a simple and label-free approach to measure simultaneously the mass uptake and viscoelastic ...

High Sensitivity Measurement of Transcription Factor-DNA Binding Affinities by Competitive Titration Using Fluorescence Microscopy

Accurate quantification of transcription factor (TF)-DNA interactions is essential for understanding the regulation of gene expression. Since existing approaches suffer from significant limitations, we have developed a new method for determining TF-DNA binding affinities with high sensitivity on a large scale. The assay relies on the established fluorescence anisotropy (FA) principle but introduces important technical improvements. First, we measure a full FA competitive titration curve in a single well by incorporating TF and a fluorescently labeled reference DNA in a porous agarose gel matrix. Unlabeled DNA oligomer is loaded on the top as a competitor and, through diffusion, forms a spatio-temporal gradient. The resulting FA gradient is then read out using a customized epifluorescence microscope setup. This improved setup greatly increases the sensitivity of FA signal detection, allowing both weak and strong binding to be reliably quantified, even for molecules of similar molecular weights. In this fashion, we can measure one titration curve per well of a multi-well plate, and through a fitting procedure, we can extract both the absolute dissociation constant (KD) and active protein concentration. By testing all single-point mutation variants of a given consensus binding sequence, we can survey the entire binding specificity landscape of a TF, typically on a single plate. The resulting position weight matrices (PWMs) outperform those derived from other methods in predicting in vivo TF occupancy. Here, we present a detailed guide for implementing HiP-FA on a conventional automated fluorescent microscope and the data analysis pipeline.

Here we present a novel method for determining binding affinities at equilibrium and in solution with high sensitivity on a large scale. This improves the quantitative analysis of transcription factor-DNA binding. The method is based on automated fluorescence anisotropy measurements in a controlled delivery system.

Accurate quantification of transcription factor (TF)-DNA interactions is essential for understanding the regulation of gene expression. Since existing ...

Analysis of Protein Folding, Transport, and Degradation in Living Cells by Radioactive Pulse Chase

Radioactive pulse-chase labeling is a powerful tool for studying the conformational maturation, the transport to their functional cellular location, and the degradation of target proteins in live cells. By using short (pulse) radiolabeling times (&lt;30 min) and tightly controlled chase times, it is possible to label only a small fraction of the total protein pool and follow its folding. When combined with nonreducing/reducing SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoprecipitation with (conformation-specific) antibodies, folding processes can be examined in great detail. This system has been used to analyze the folding of proteins with a huge variation in properties such as soluble proteins, single and multi-pass transmembrane proteins, heavily N- and O-glycosylated proteins, and proteins with and without extensive disulfide bonding. Pulse-chase methods are the basis of kinetic studies into a range of additional features, including co- and posttranslational modifications, oligomerization, and polymerization, essentially allowing the analysis of a protein from birth to death. Pulse-chase studies on protein folding are complementary with other biochemical and biophysical methods for studying proteins in vitro by providing increased temporal resolution and physiological information. The methods as described within this paper are adapted easily to study the folding of almost any protein that can be expressed in mammalian or insect-cell systems.

Here we describe a protocol for a general pulse-chase method that allows the kinetic analysis of folding, transport, and degradation of proteins to be followed in live cells.

Radioactive pulse-chase labeling is a powerful tool for studying the conformational maturation, the transport to their functional cellular location, and ...

Absolute Quantification of Cell-Free Protein Synthesis Metabolism by Reversed-Phase Liquid Chromatography-Mass Spectrometry

Cell-free protein synthesis (CFPS) is an emerging technology in systems and synthetic biology for the in vitro production of proteins. However, if CFPS is going to move beyond the laboratory and become a widespread and standard just in time manufacturing technology, we must understand the performance limits of these systems. Toward this question, we developed a robust protocol to quantify 40 compounds involved in glycolysis, the pentose phosphate pathway, the tricarboxylic acid cycle, energy metabolism and cofactor regeneration in CFPS reactions. The method uses internal standards tagged with 13C-aniline, while compounds in the sample are derivatized with 12C-aniline. The internal standards and sample were mixed and analyzed by reversed-phase liquid chromatography-mass spectrometry (LC/MS). The co-elution of compounds eliminated ion suppression, allowing the accurate quantification of metabolite concentrations over 2-3 orders of magnitude where the average correlation coefficient was 0.988. Five of the forty compounds were untagged with aniline, however, they were still detected in the CFPS sample and quantified with a standard curve method. The chromatographic run takes approximately 10 min to complete. Taken together, we developed a fast, robust method to separate and accurately quantify 40 compounds involved in CFPS in a single LC/MS run. The method is a comprehensive and accurate approach to characterize cell-free metabolism, so that ultimately, we can understand and improve the yield, productivity and energy efficiency of cell-free systems.

Here, we present a robust protocol to quantify 40 compounds involved in central carbon and energy metabolism in cell-free protein synthesis reactions. The cell-free synthesis mixture is derivatized with aniline for effective separation using reversed-phase liquid chromatography and then quantified by mass spectrometry using isotopically labelled internal standards.

Cell-free protein synthesis (CFPS) is an emerging technology in systems and synthetic biology for the in vitro production of proteins. However, if CFPS is ...

Creating Highly Specific Chemically Induced Protein Dimerization Systems by Stepwise Phage Selection of a Combinatorial Single-Domain Antibody Library

Protein dimerization events that occur only in the presence of a small-molecule ligand enable the development of small-molecule biosensors for the dissection and manipulation of biological pathways. Currently, only a limited number of chemically induced dimerization (CID) systems exist and engineering new ones with desired sensitivity and selectivity for specific small-molecule ligands remains a challenge in the field of protein engineering. We here describe a high throughput screening method, combinatorial binders-enabled selection of CID (COMBINES-CID), for the de novo engineering of CID systems applicable to a large variety of ligands. This method uses the two-step selection of a phage-displayed combinatorial nanobody library to obtain 1) &#34;anchor binders&#34; that first bind to a ligand of interest and then 2) &#34;dimerization binders&#34; that only bind to anchor binder-ligand complexes. To select anchor binders, a combinatorial library of over 109 complementarity-determining region (CDR)-randomized nanobodies is screened with a biotinylated ligand and hits are validated with the unlabeled ligand by bio-layer interferometry (BLI). To obtain dimerization binders, the nanobody library is screened with anchor binder-ligand complexes as targets for positive screening and the unbound anchor binders for negative screening. COMBINES-CID is broadly applicable to select CID binders with other immunoglobulin, non-immunoglobulin, or computationally designed scaffolds to create biosensors for in vitro and in vivo detection of drugs, metabolites, signaling molecules, etc.

Creating chemically induced protein dimerization systems with desired affinity and specificity for any given small molecule ligand would have many biological sensing and actuation applications. Here, we describe an efficient, generalizable method for de novo engineering of chemically induced dimerization systems via the stepwise selection of a phage-displayed combinatorial single-domain antibody library.

Protein dimerization events that occur only in the presence of a small-molecule ligand enable the development of small-molecule biosensors for the ...

OaAEP1-Mediated Enzymatic Synthesis and Immobilization of Polymerized Protein for Single-Molecule Force Spectroscopy

Chemical and bio-conjugation techniques have been developed rapidly in recent years and allow the building of protein polymers. However, a controlled protein polymerization process is always a challenge. Here, we have developed an enzymatic methodology for constructing polymerized protein step by step in a rationally-controlled sequence. In this method, the C-terminus of a protein monomer is NGL for protein conjugation using OaAEP1 (Oldenlandia affinis asparaginyl endopeptidases) 1) while the N-terminus was a cleavable TEV (tobacco etch virus) cleavage site plus an L (ENLYFQ/GL) for temporary N-terminal protecting. Consequently, OaAEP1 was able to add only one protein monomer at a time, and then the TEV protease cleaved the N-terminus between Q and G to expose the NH2-Gly-Leu. Then the unit is ready for next OaAEP1 ligation. The engineered polyprotein is examined by unfolding individual protein domain using atomic force microscopy-based single-molecule force spectroscopy (AFM-SMFS). Therefore, this study provides a useful strategy for polyprotein engineering and immobilization.

Here, we present a protocol to conjugate protein monomer by enzymes forming protein polymer with a controlled sequence and immobilize it on the surface for single-molecule force spectroscopy studies.

Chemical and bio-conjugation techniques have been developed rapidly in recent years and allow the building of protein polymers. However, a controlled ...

Native Cell Membrane Nanoparticles System for Membrane Protein-Protein Interaction Analysis

Protein-protein interactions in cell membrane systems play crucial roles in a wide range of biological processes- from cell-to-cell interactions to signal transduction; from sensing environmental signals to biological response; from metabolic regulation to developmental control. Accurate structural information of protein-protein interactions is crucial for understanding the molecular mechanisms of membrane protein complexes and for the design of highly specific molecules to modulate these proteins. Many in vivo and in vitro approaches have been developed for the detection and analysis of protein-protein interactions. Among them the structural biology approach is unique in that it can provide direct structural information of protein-protein interactions at the atomic level. However, current membrane protein structural biology is still largely limited to detergent-based methods. The major drawback of detergent-based methods is that they often dissociate or denature membrane protein complexes once their native lipid bilayer environment is removed by detergent molecules. We have been developing a&#160;native cell membrane nanoparticle system for membrane protein structural biology. Here, we demonstrate the use of this system in the analysis of protein-protein interactions on the cell membrane with a case study of the oligomeric state of AcrB.

Presented here is a protocol for the determination of oligomeric state of membrane proteins that utilizes a native cell membrane nanoparticle system in conjunction with electron microscopy.

Protein-protein interactions in cell membrane systems play crucial roles in a wide range of biological processes- from cell-to-cell interactions to signal ...

Cell-Free Production of Proteoliposomes for Functional Analysis and Antibody Development Targeting Membrane Proteins

Membrane proteins play essential roles in a variety of cellular processes and perform vital functions. Membrane proteins are medically important in drug discovery because they are the targets of more than half of all drugs. An obstacle to conducting biochemical, biophysical, and structural studies of membrane proteins as well as antibody development has been the difficulty in producing large amounts of high-quality membrane protein with correct conformation and activity. Here we describe a &#8220;bilayer-dialysis method&#8221; using a wheat germ cell-free system, liposomes, and dialysis cups to efficiently synthesize membrane proteins and prepare purified proteoliposomes in a short time with a high success rate. Membrane proteins can be produced as much as in several milligrams, such as GPCRs, ion channels, transporters, and tetraspanins. This cell-free method contributes to reducing the time, cost and effort for preparing high-quality proteoliposomes, and provides suitable means for functional analysis of membrane proteins, drug targets screening, and antibody development.

This protocol describes an efficient cell-free method for production of high-quality proteoliposome by bilayer-dialysis method using wheat cell-free system and liposomes. This method provides suitable means for functional analysis of membrane proteins, drug targets screening, and antibody development.

Membrane proteins play essential roles in a variety of cellular processes and perform vital functions. Membrane proteins are medically important in drug ...