Webinar recap: Frank Bernhard’s Cell-Free Workflow for Cryo-EM–Ready Membrane Protein Complexes

Webinar recap: Frank Bernhard’s Cell-Free Workflow for Cryo-EM–Ready Membrane Protein Complexes

When dealing with complex G protein-coupled receptors (GPCRs), traditional cell-based expression of membrane proteins often results in toxicity, aggregation, or the need for heavy engineering just to get the protein to the membrane. Once in the membrane, detergent extraction removes the critical lipids GPCRs required for stability and function.

 

Dr. Bernhard’s cell-free workflow removes these biological bottlenecks by using a processed E. coli lysate where the cellular machinery is accessible, allowing researchers to tune the environment to suit the protein. To hear about the workflow in more detail, sign up for the on-demand webinar.

The core idea: a controllable, “open” expression system

Dr. Bernhard emphasized one defining advantage of cell-free expression: the reaction is open, meaning you can add stabilizing compounds, ligands, cofactors, or other components directly into the translation environment, at the time point and concentration you choose, without worrying about cell viability or metabolism.

The “open” reaction environment

The core of Dr. Bernhard’s workflow is a T7-based transcription-translation setup and processed, tunable E. coli lysates housed in a two-compartment reactor (2C) (Figure 1)1. This separates the reaction mix from the feeding mix via a semi-permeable membrane, allowing for continuous supply of precursors and removal of byproducts. Dr. Bernhard uses this continuous exchange cell-free setup for high-yield protein production in reaction volumes ranging from 30 µL (for screening) to 10 mL (for cryo-EM preparation) in few hours or overnight.   Pro Tip: For disulfide-rich proteins, the redox environment (DTT, GSH/GSSG) can be chemically tuned—something impossible in live E. coli.

Figure 1. The 2C system for cell-free protein synthesis. The inner chamber is the reaction compartment (light blue) while the surrounding compartment (blue) is the feeding compartment.

• Pro Tip: If yields are low, the culprit is often translation initiation. Adding 1–6 AT-rich codons as “expression tag” immediately downstream of the start codon can boost expression by orders of magnitude.

While cell-free systems require detergent-based solubilization, Dr. Bernhard advocates for Lipid-based Cell-Free (L-CF) expression. By adding pre-assembled nanodiscs (NDs), or other nanoparticles, directly into the reaction, the nascent protein inserts co-translationally into a defined lipid bilayer (Figure 2).   Why it works: This bypasses limiting translocon and targeting machineries. The interface between the scaffold protein and the lipid provides a non-specific entry site for membrane proteins such as GPCRs. Lipid Choice: While there are many nanodisc compositions to choose from, Dr. Bernhard recommends to start with negatively charged lipids Dimyristoyl phosphatidylglycerol (DMPG), Dioleoylphosphatidylglycerol (DOPG), and Palmitoyloleoyl-phosphatidylglycerol (POPG). “These usually give you instantly good insertion and folding results.”

Figure 2. In the L-CF mode, the ribosome synthesizes the protein which co-translationally inserts into pre-formed nanodiscs.

Quality Control: The “NSEC” Method

A major pitfall in membrane protein expression is assuming that expression of the soluble/fluorescent protein often used as a readout for protein folding equals functional protein. Dr. Bernhard’s data shows that green fluorescent protein (GFP) fluorescence often does not correlate with ligand binding activity.

Instead, the team uses Nanoparticle Size Exclusion Chromatography (NSEC) where proteins elute based on their folding outcomes.

 

• The “1.5 Peak”: Empty nanodiscs and properly loaded nanodiscs elute at the same volume (approx 1.5 mL with their SEC setup).
• The Aggregation Peak: Unfolded or aggregated proteins attached to the outside of the disc elute earlier due to their larger hydrodynamic radius.
• The Strategy: Optimize the reaction (e.g. screening lipids, ligands) to maximize the “1.5 peak” and purify. This peak consistently contains the active fraction, allowing for fast quality control and optimization without the need for complex functional assays.

Case studies: full-length GPCRs and cryo-EM–ready complexes

A standout theme was how cell-free workflows can enable full-length GPCRs without the heavy engineering (large inserts, truncations) often used in cell-based expression for stability. Dr. Bernhard shared examples of cryo-EM structures of human GPCR-G protein complexes purified from cell-free expression including:

• Histamine 2 receptor (H2R)/Gs complex²
• β1-adrenergic receptor (β1AR)/Gs³

A further advantage of this workflow is best illustrated by β1AR. Previous structural studies relied on a truncated turkey β1AR derivatives expressed in insect cells. Using the cell-free workflow, Dr. Bernhard’s team produced the full-length human β1AR.

 

• The Result: The cryo-EM structure (3.3 Å) revealed the complete receptor, including N- and C-terminal domains and the full Internal Loop 3 (ICL3).
• The Impact: This revealed new interaction clusters and tighter G-protein coupling compared to previous truncated turkey models, providing a more accurate template for drug discovery.

 

 

 

Cryo-EM structure of a cell-free synthesized full-length human β1-adrenergic receptor in complex with Gs – Structure (Cell Press) and Cryo-EM structure of cell-free synthesized human histamine 2 receptor/Gs complex in nanodisc environment – Nature Communications (Nature publishing) reproduced via creative commons license CC by 4.0 licence.

Case study: Nanotransfer of GPCRs into living cells

Finally, Dr. Bernhard introduced “nanotransfer”, a method to fuse membrane protein loaded nanodiscs with the membranes of living mammalian cells. This allows cell-free synthesized proteins to be transferred into live cells to study their function (e.g., homo- and hetero-dimerization of FFAR₂/FFAR₃) in a native cellular context⁴. This bridges the gap between high-resolution in vitro structural and functional analysis and in vivo biological function.

 

“It’s a fast and easy technique,” said Dr. Bernhard. “You just combine cells and your purified nanoparticle/membrane protein complexes in a tube.” He also noted that this technique provides more uniform membrane protein distribution compared to transfection, and unlike transfection, it is suitable for primary cells.

Conclusions

Dr. Bernhard’s work demonstrates that cell-free protein synthesis is no longer just an alternative expression method, it is a robust, front-line platform for generating high-quality membrane proteins for structural biology. This workflow enables the study of full-length, wild-type human receptors that were previously much more difficult to access

These principles perfectly align with Nuclera’s goal to remove the guesswork from membrane protein expression and purification. By pairing the power of pre-assembled, membrane insertion optimized nanodiscs with the automated multiplex screening of the eProtein Discovery™ system, researchers can rapidly identify optimal stabilization conditions and scale up to assay-ready, purified GPCRs in just 48 hours.

To get the full details of Dr. Bernhard’s cell-free protein expression workflow and expert tips, view the full webinar on-demand.

Q&A

Answers by Dr. Bernhard

Q. When screening for nanodiscs, is the size of the disc or the phospholipid composition more important?

A. Both are important, but stability often dictates the size choice. Dr. Bernhard prefers the MSP1E3D1 scaffold, which provides an ideal balance of stability and insertion space. While he notes that smaller discs may offer higher resolution for cryo-EM, larger nanodiscs (approx. 15 nm) can become unstable in cell-free reactions.

Regarding lipids, the optimal composition varies greatly based on the specific research need. For example, DOPG is considered the proven standard for cryo-EM applications, while complex, cholesterol-dependent GPCRs may require POPC/POPS/Cholesterol blends for essential functionality. Because identifying this optimal environment is so critical, screening a pre-assembled panel of nanodiscs with diverse lipid geometries is the most efficient way to maximize yield and activity for your specific target.

 

Q. How reproducible is the yield and the quality of different protein batches produced using Dr. Bernhard’s protocol?

A. If you maintain the strict quality of your ingredients, reproducibility is excellent. Once a protocol is established, Dr. Bernhard observes consistent yield and quality across batches. To minimize variability between users, he recommends running a positive control (like GFP) to benchmark the system (aiming for ~3 mg/mL) and centralizing stock management so one technician prepares the basic solutions

 

Q. How successful is co-expression with the nanodisc?

A. While co-expression of the scaffold and target protein is possible, comparative studies in Dr. Bernhard’s lab show that using pre-formed nanodiscs consistently yields better results. Co-expression suffers from a lack of control over stoichiometry, meaning you cannot precisely dictate how the scaffold and target assemble. Overcoming this requires a significant time investment to fine-tune DNA template ratios and lipid concentrations.

This is why standardized, pre-assembled nanodiscs are preferable for structural biology workflows. They allow researchers to bypass the trial-and-error of co-expression, ensuring robust data quality and accelerating the path to downstream applications.

 

Q. After reconstitution of my membrane protein into nanodiscs, is it better to separate and purify assembled proteins using SEC or affinity chromatography?

A. Dr. Bernhard mentioned that affinity chromatography is usually sufficient for removing empty pre-assembled nanodiscs and most lysate proteins, but subsequent size exclusion chromatography is essential for evaluating the overall quality and homogeneity of your sample. Dr. Bernhard uses SEC to remove misfolded or aggregated protein from monomeric, active protein fractions.

 

Q. Does lipid composition of the nanodiscs affect the orientation in a cryo-EM grid?

A. No evidence, Dr. Bernhard has found that the particles analyzed so far exhibit a completely random orientation regardless of the lipid composition used, which is an ideal scenario for high-quality cryo-EM data collection. However, while orientation remains random, certain lipid compositions do act as gold standards for membrane protein folding and overall complex stability during cryo-EM. Screening a panel of nanodiscs upstream ensures you are taking the most stable, highest-resolution candidate into your downstream structural studies.

 

Q. Can you directly apply the final extract on EM grids or do you have to run some purification step?

A. A purification step is absolutely required to isolate your target complex from the rest of the lysate components, such as ribosomes and enzymes. Dr. Bernhard uses a rapid four-step workflow: first, he transfers the cell-free reaction directly to affinity purification, then he uses SEC to purify the specific peak corresponding to the target protein/nanodisc complex; next, he concentrates that specific fraction; and finally, he applies the purified, concentrated sample to the Cryo-EM grid. He notes that this process is quite streamlined, with overnight cell-free expression and the whole purification/grid formation process completed the next day by the afternoon.

 

References

1. Schwarz, D. et al. Preparative scale expression of membrane proteins in Escherichia coli-based continuous exchange cell-free systems. Nat. Protoc. 2, 2945–2957 (2007).
2. Köck, Z. et al. Cryo-EM structure of cell-free synthesized human histamine 2 receptor/Gs complex in nanodisc environment. Nat. Commun. 15, 1831 (2024).
3. Merino, F. et al. Cryo-EM structure of a cell-free synthesized full-length human β1-adrenergic receptor in complex with Gs. Structure 33, 1867–1877.e5 (2025).
4. Umbach, S. et al. Transfer mechanism of cell-free synthesized membrane proteins into mammalian cells. Front. Bioeng. Biotechnol. 10, 906295 (2022).