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Method Article

Reconstitution of the Bacterial Glutamate Receptor Channel by Encapsulation of a Cell-Free Expression System

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DOI:

10.3791/66595

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March 8th, 2024

 ,  , 
1Neuroscience Program, University of Michigan, Ann Arbor, 2Cellular and Molecular Biology Program, University of Michigan, Ann Arbor, 3Department of Mechanical Engineering, University of Michigan, Ann Arbor, 4Department of Biomedical Engineering, University of Michigan, Ann Arbor, 5Department of Biophysics, University of Michigan, Ann Arbor

* These authors contributed equally

In This Article

Summary

This protocol describes the inverted emulsion method used to encapsulate a cell-free expression (CFE) system within a giant unilamellar vesicle (GUV) for the investigation of the synthesis and incorporation of a model membrane protein into the lipid bilayer.

Abstract

Cell-free expression (CFE) systems are powerful tools in synthetic biology that allow biomimicry of cellular functions like biosensing and energy regeneration in synthetic cells. Reconstruction of a wide range of cellular processes, however, requires successful reconstitution of membrane proteins into the membrane of synthetic cells. While the expression of soluble proteins is usually successful in common CFE systems, the reconstitution of membrane proteins in lipid bilayers of synthetic cells has proven to be challenging. Here, a method for reconstitution of a model membrane protein, bacterial glutamate receptor (GluR0), in giant unilamellar vesicles (GUVs) as model synthetic cells based on encapsulation and incubation of the CFE reaction inside synthetic cells is demonstrated. Utilizing this platform, the effect of substituting the N-terminal signal peptide of GluR0 with proteorhodopsin signal peptide on successful cotranslational translocation of GluR0 into membranes of hybrid GUVs is demonstrated. This method provides a robust procedure that will allow cell-free reconstitution of various membrane proteins in synthetic cells.

Introduction

Bottom-up synthetic biology has gained increasing interest over the past decade as an emerging field with numerous potential applications in bioengineering, drug delivery, and regenerative medicine1,2. The development of synthetic cells as a cornerstone of bottom-up synthetic biology, in particular, has attracted a wide range of scientific communities due to the promising applications of synthetic cells as well as their cell-like physical and biochemical properties that facilitate in vitro biophysical studies3,4,5,6. Synthetic cells are often engineered in cell-sized giant unilamellar vesicles (GUVs) in which different biological processes are recreated. Reconstitution of cell cytoskeleton7,8, light-dependent energy regeneration9, cellular communication10,11, and biosensing12 are examples of efforts made to reconstruct cell-like behaviors in synthetic cells.

While some cellular processes rely on soluble proteins, many characteristics of natural cells, such as sensing and communication, often utilize membrane proteins, including ion channels, receptors, and transporters. A major challenge in synthetic cell development is the reconstitution of membrane proteins. Although traditional methods of membrane protein reconstitution in lipid bilayers rely on detergent-mediated purification, such methods are laborious, ineffective for proteins that are toxic to the expression host, or are often not suited for membrane protein reconstitution in GUVs13.

An alternative method for protein expression is cell-free expression (CFE) systems. CFE systems have been a powerful tool in synthetic biology that allows in vitro expression of various proteins using either cell lysate or purified transcription-translation machinery14. CFE systems can also be encapsulated in GUVs, thus allowing compartmentalized protein synthesis reactions that can be programmed for various applications, such as the creation of light-harvesting synthetic cells9 or mechanosensitive biosensors15,16. Analogous to recombinant protein expression methods, membrane protein expression is challenging in CFE systems17. Aggregation, misfolding, and lack of post-translational modification in CFE systems are major bottlenecks that hinder successful membrane protein synthesis using CFE systems. The difficulty of bottom-up membrane protein reconstitution using CFE systems is due in part to the absence of a complex membrane protein biogenesis pathway that relies on signal peptides, signal recognition particles, translocons, and chaperoning molecules. However, recently, multiple studies have suggested that the presence of membranous structures such as microsomes or liposomes during translation promotes successful membrane protein expression18,19,20,21. Additionally, Eaglesfield et al. and Steinküher et al. have found that the inclusion of specific hydrophobic domains known as signal peptides in the N-terminus of the membrane protein can significantly improve its expression22,23. Altogether, these studies suggest that the challenge of membrane protein reconstitution in synthetic cells can be overcome if the protein translation occurs in the presence of the GUV membrane and if proper N-terminal signal peptide is utilized.

Here, a protocol for encapsulation of the protein synthesis using recombinant elements (PURE) CFE reactions for membrane protein reconstitution in GUVs is presented. Bacterial glutamate receptor24 (GluR0) is selected as the model membrane protein, and the effect of its N-terminal signal peptide on its membrane reconstitution is studied. The effect of proteorhodopsin signal peptide, which was shown to improve membrane protein reconstitution efficiency by Eaglesfield et al.22, is investigated by constructing a mutated variant of GluR0 denoted as PRSP-GluR0 and its expression and membrane localization with wild-type GluR0 (referred to as WT-GluR0 hereafter) that harbors its native signal peptide is compared. This protocol is based on the inverted emulsion method25 with modifications that make it robust for CFE encapsulation. In the presented method, the CFE reactions are first emulsified using a lipid-in-oil solution that generates micron-sized droplets that contain the CFE system and are stabilized by the lipid monolayer. The emulsion droplets are then layered on top of an oil-water interface that is saturated with another lipid monolayer. The emulsion droplets are then forced to travel across the oil-water interface via centrifugal force. Through this process, the droplets obtain another monolayer, thus generating a bilayer lipid vesicle. The GUVs containing the CFE reaction are then incubated, during which the membrane protein is expressed and incorporated into the GUV membrane. Although this protocol is specified for cell-free expression of GluR0, it can be used for cell-free synthesis of other membrane proteins or different synthetic cell applications such as cytoskeleton reconstitution or membrane fusion studies26.

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Protocol

The reagents and equipment utilized for this study are provided in the Table of Materials.

1. Bulk CFE reactions in the presence of small unilamellar vesicles (SUVs)

  1. SUV preparation
    NOTE: This step needs to be performed in a fume hood following the safety instructions for working with chloroform.
    1. Prepare 5 mM 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) SUVs in a glass vial by transferring 76 µL of 25 mg/mL POPC stock solution dissolved in chloroform.
    2. While gently rotating the glass vial, blow a gentle stream of argon into the vial to form a film of dried lipids at the bottom. Next, transfer the glass vial to a desiccator with its cap loosely screwed to evaporate excess chloroform.
    3. Keep the glass vial in the desiccator for 1 h. Then, add 0.5 mL ultra-pure deionized water to dissolve the lipid film and vortex for approximately 2 min.
    4. Set up a mini-extrusion apparatus by soaking two filter supports in deionized water and placing them on each of the internal membrane supports. Then, soak a 100 nm polycarbonate filter and place it in between the two internal membrane supports held together by the extruder outer casing and the retainer nut. Place this setup in the extruder stand.
    5. Flush two 1 mL gas-tight syringes 3 times with ultra-pure deionized water.
    6. Load the sample of lipid-water mix into one of the 1 mL gas-tight syringes and place it in one end of the mini extruder using the swing arm clips to hold the syringe in place. Insert the second syringe into the other end of the mini-extruder and make sure it is fully depressed.
      NOTE: When loading the syringe with the lipid-water mix, ensure there is no air in the syringe before passing it through the mini-extruder.
    7. Gently pass the lipid-water mix from the original syringe to the empty syringe through the mini-extruder apparatus. Repeat this step 11 times to form SUVs. Transfer the SUVs to a 1.5 mL microcentrifuge tube.
      NOTE: The SUV solution can be stored at 4 °C for up to 2 weeks.
  2. CFE reaction assembly
    1. Assemble the CFE reaction by following the cell-free expression protocol provided by the manufacturer with slight modifications detailed in the following.
    2. Mix 10 µL of Solution 1 (containing amino acids, NTPs, tRNAs and substrates for enzymes, and necessary buffer), 1 µL of Solution 2 (proteins in 20% glycerol), 2 µL of Solution 3 (ribosome (20 µM)), appropriate amount of the DNA encoding for soluble sfGFP-sfCherry(1-10)27 (referred to as soluble sfGFP hereafter), WT-GluR0-sfGFP, or PRSP-GluR0-sfGFP (10-60 ng/1000 base pairs), 1 µL murine RNAse inhibitor, and 4 µL of 5 mM SUV solution for membrane proteins or 4 µL water for soluble proteins.
    3. Bring the reaction's final volume to 20 µL by adding ultra-pure deionized water.
      NOTE: When assembling the CFE system, all components must be kept on ice. All materials are temperature-sensitive and can degrade if they reach room temperature.
  3. CFE reaction incubation and monitoring
    1. Transfer the CFE solution to a 96-well conical V-bottom plate. To prevent evaporation during the course of the reaction, cover the plate using a sealing film.
    2. Incubate the plate at 37 °C in a plate reader for 4-5 h while monitoring the CFE reaction by measuring the GFP signal with a gain of 100 at 488 nm/528 nm excitation/emission wavelengths every 2 min.

2. CFE reactions encapsulated in GUVs

  1. Preparation of GUV outer buffer solution
    1. In a 1.5 mL microcentrifuge tube, mix 1.5 µL of 1 M spermidine, 37.5 µL of 100 mM ATP, 25 µL of 100 mM GTP, 12.5 µL of 100 mM CTP, 12.5 µL of 100 mM UTP, 25 µL of 1 M creatine phosphate, 18 µL of 1 M magnesium acetate, 93.33 µL of 3 M potassium glutamate, 50 µL of 1 M HEPES KOH (pH 7.4), 1.15 µL of 332 mM folinic acid, 100 µL of 2 M glucose, and 50 µL from stock solution of 6 mM mixture of each of the 20 amino acids (prepared by following the protocol described by Sun et al.28).
    2. Bring the solution's final volume to 1 mL by adding ultra-purified deionized water.
      NOTE: All components must be kept on ice. Add the amino acid mixture at the end to avoid amino acid depletion28. The solution can be aliquoted in 330 µL aliquots and stored at -20 °C until use.
  2. Preparation of the lipid-in-oil mixture
    NOTE: this step needs to be performed in a fume hood following the safety instructions for working with chloroform.
    1. In a fume hood, mix 17.3 µL of 25 mg/mL POPC stock solution and 1.08 µL of 50 mg/mL poly(butadiene)-b-poly(ethylene oxide) (PEO-b-PBD) copolymer in a 15 mL glass vial.
      NOTE: The final lipid-in-oil solution contains 0.5 mM lipid with 95% and 5% POPC and PEO-b-PBD, respectively. PEO-b-PBD was used to enhance membrane stability during protein expression but was kept at a low molar ratio to reduce the copolymer's tendency to aggregate into micelles separate from lipid molecules29,30.
    2. Carefully blow a gentle stream of argon gas into the glass vial while rotating the vial to evaporate the chloroform.
    3. Pipette 1.2 mL of light mineral oil into the 15 mL glass vial containing the dried lipids.
    4. Mix the lipids and oil by vortexing at maximum speed for 10-20 s. The dissolved lipid-copolymer mix will look cloudy.
    5. To ensure that all possible lipid aggregates in the oil are fully dissolved and dispersed throughout the oil, place the glass vial in an oven at around 50 °C for 20 min before vortexing for an additional 10-20 s at maximum speed.
  3. CFE reaction assembly and encapsulation (Figure 1 summarizes the following steps).
    1. Prepare 300 µL of the GUV outer solution in a 1.5 mL microcentrifuge tube by mixing 270 µL of CFE outer buffer solution prepared in step 2.1, 15 µL of 5 M NaCl, and 15 µL of 4.5 M KCl.
      NOTE: At this step, add 0.45 µL of 1 M 1,4-dithiothreitol (DTT) to the GUV outer solution. The purpose of adding NaCl and KCl is to adjust the outer solution's osmolality to match it with the inner CFE solution. The exact volume of NaCl and KCl depends on the desired osmolality adjustment. One can add either NaCl or KCl or both to adjust the osmolality.
    2. Gently pipette 300 µL of lipid-in-oil mixture on top of the GUV outer solution.
      NOTE: When adding the lipid-oil mix, it is important that the oil does not mix with the aqueous outer solution. After the addition, there should be a visible interface between the lipid-in-oil mixture and the GUV outer solution.
    3. Incubate the oil-water interface at room temperature for 2 h to allow the lipid monolayer to form and stabilize at the interface.
    4. Meanwhile, follow section 1.2.1 to assemble a CFE reaction containing the plasmid DNA encoding the membrane protein variants or soluble GFP. Replace the 4 µL of 5 mM SUV solution or water with 4 µL of 1 M sucrose. This reaction will be the GUV inner solution.
      NOTE: An osmometer was used to measure the osmolality of the inner and outer solutions. The osmolality of the outer solution was then adjusted accordingly by the addition of NaCl or water. The osmolality of the CFE reaction is typically around 1600 mOsm/Kg. Adding sucrose to the reaction increases the inner solution density, thus allowing the vesicles to travel across the oil-water interface during the centrifugation step. An alternative to sucrose is the Opti-Prep density gradient solution.
    5. Add 600 µL of lipid-oil mixture to the microcentrifuge tube containing the CFE reaction and pipette up and down vigorously for ~1 min to emulsify the reaction in lipid-in-oil solution and form the lipid monolayer around synthetic cells.
      NOTE: The final solution should not have any bubbles and should look opaque.
    6. Gently pipette the inner solution emulsion on top of the oil layer in the 1.5 mL microcentrifuge tube where the oil-water interface was set up.
      NOTE: Be careful not to disturb or destabilize the interface.
    7. Centrifuge for 10 min at 2,000 x g at 4 °C.
      NOTE: The centrifugation speed was optimized for this protocol. Adir et al.31 reported a different centrifugation speed.
    8. Once the centrifugation is over, carefully remove the excess oil and outer solution from the microcentrifuge tube using a pipettor. Remove the outer solution until the remaining volume is around 100 µL.
      NOTE: A pellet of GUVs is usually visible at the bottom of the microcentrifuge tube. However, a lack of a visible pellet does not necessarily mean no GUV yield. Instead of using a pipettor, the excess oil on top of the outer solution can be removed by aspiration. It is critical to ensure the lipid-in-oil solution is completely removed. Oil contamination in GUV solution can cause low-quality images.
    9. Resuspend the GUV pellet in the remaining 100 µL solution by gently pipetting up and down. Next, transfer the GUV solution to a clean 96 well clear flat bottom plate to incubate.

3. Encapsulated CFE reaction incubation and imaging

  1. Cover the plate using a sealing film to prevent evaporation. Incubate the plate at 37 °C for 5-6 h. One can use a plate reader and follow step 1.3.2 to prepare the plate reader for the incubation step.
  2. Once the incubation is over, place the 96 well plate on the imaging stage of an inverted microscope equipped with an EMCCD camera (or a sCMOS camera), DAQ-MX controlled laser (or an integrated laser combiner system), and a CSU-X1 spinning disk confocal (or a laser scanning confocal). Focus on any ROI containing GUVs and capture images at an excitation wavelength of 488 nm using a Plan-Apochromat 60 x/1.4 NA objective.
  3. Save images of GUVs in .tiff format.
  4. Open the images in an image processing software (e.g., ImageJ or Fiji). Open the Brightness/Contrast setting panel. Adjust the brightness and contrast to appropriate settings that make fluorescent proteins visible.
  5. If the goal is to compare the signal intensity of different expressed proteins, first stack individual images of GUVs containing different proteins using Images to Stack panel located under Image > Stacks submenu. Then, adjust the brightness and contrast of all images using the Brightness/Contrast panel.

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Results

Prior to encapsulation of the CFE reactions, two variants of GluR0-sfGFP harboring native and proteorhodopsin signal peptides (signal peptide sequences are presented in Supplementary Table 1), and the soluble sfGFP were individually expressed in bulk reactions, and their expression was monitored by detecting the sfGFP signal using a plate reader (Figure 2A). Membrane proteins were expressed in the absence or presence of 100 nm SUVs. Additionally, using a cal...

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Discussion

Virtually any cellular process that depends on the transfer of molecules or information across the cell membrane, like cell signaling or cell excitation, requires membrane proteins. Thus, the reconstitution of membrane proteins has become the main bottleneck in realizing various synthetic cell designs for different applications. Traditional detergent-mediated reconstitution of membrane proteins in biological membranes requires GUV generation methods such as gentle swelling or electroformation. Swelling approaches usually...

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Disclosures

The authors declare no conflicts of interest.

Acknowledgements

APL acknowledges support from the National Science Foundation (EF1935265), the National Institutes of Health (R01-EB030031 and R21-AR080363), and the Army Research Office (80523-BB)

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
100 nm polycarbonate filterSTERLITECH1270193
96 Well Clear Bottom PlateThermoFisher Scientific165305
BioTek Synergy H1M Hybrid Multi-Mode ReaderAgilent11-120-533
Creatine phosphateMillipore Sigma10621714001
CSU-X1 Confocal Scanner UnitYokogawaCSU-X1 
Density gradient medium (Optiprep)Millipore SigmaD1556Optional to switch with sucrose in inner solution
Filter supportsAvanti610014
Fisherbrand microtubes (1.5 mL)Fisher Scientific05-408-129 
Folinic acid calcium salt hydrateMillipore SigmaF7878
GlucoseMillipore Sigma158968
HEPESMillipore SigmaH3375
iXon X3 camera AndorDU-897E-CS0 
L-Glutamic acid potassium salt monohydrateMillipore SigmaG1501
Light mineral oilMillipore SigmaM5904
Magnesium acetate tetrahydrate Millipore SigmaM5661
Mini-extruder kit (including syringe holder and extruder stand)Avanti610020
Olympus IX81 Inverted Microscope OlympusIX21
Olympus PlanApo N 60x Oil Microscope Objective Olympus1-U2B933 
PEO-b-PBDPolymer SourceP41745-BdEO
pET28b-PRSP-GluR0-sfGFP plasmid DNAHomemadeN/A
pET28b-sfGFP-sfCherry(1-10) plasmid DNAHomemadeN/A
pET28b-WT-GluR0-sfGFP plasmid DNAHomemadeN/A
POPC lipid in chloroform Avanti850457C
Potassium chlorideMillipore SigmaP9541
PUREfrex 2.0Cosmo Bio USAGFK-PF201
Ribonucleotide Solution SetNew England BioLabsN0450
RNase Inhibitor, MurineNew England BioLabsM0314S
RTS Amino Acid SamplerBiotechrabbitBR1401801
Sodium chlorideMillipore SigmaS9888
SpermidineMillipore SigmaS2626
SucroseMillipore SigmaS0389
VAPRO Vapor Pressure Osmometer Model 5600ELITechGroupVAPRO 5600

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