Advancing Membrane Protein Research With Cell-Free Expression Systems

Since we are talking specifically about membrane proteins, I think the key bottleneck would be expression yield; membrane proteins express poorly in conventional systems due to toxicity or misfolding.

That brings me to the second point, which is protein misfolding and aggregation. Because proper folding often requires native lipid environments – and when we are unable to provide that, the protein misbehaves – we can add other stabilizing agents for structural stability and chaperones.

Membrane proteins also face challenges because of difficulties in purification strategies. To maintain structural integrity during the extraction and purification, the entire workflow is challenging.

Next is functional instability. Even though the protein has gone through the entire process of expression purification, it's not a high success rate that the protein you are getting is functionally active as well.

In terms of emerging solutions, cell-free expression systems, such as those provided by Nuclera, are available. These enable a more controlled environment and cause translational insertion of the membrane proteins into lipid mimetics, such as nanodiscs. This will improve folding and functionality, because various synthetic biology approaches or construct designs can be done so that when you have a challenging protein from the beginning, you can address in terms of what domains or what disordered regions you want to eliminate. Then you can customize the construct design. You can tweak your expression conditions by trying different nanodiscs.

For example, in Nuclera’s system, you can do a comprehensive analysis of different expression conditions using different constructs, customized constructs and customized cell-free blends with detergents or nanodiscs to come together with a workflow that's best for your protein. 

I think the key components for working with membrane proteins are detergents and nanodiscs. They are essential to stabilize the proteins, and they bring the protein in a near-native state for biophysical assays.

A future upcoming trend is smarter nanodiscs with tunable compositions. You can use different membrane scaffold proteins, different cholesterol or membrane-associated cofactors. You can do high-throughput compatibility, where in systems such as those using digital microfluidics, you can go for a screening of different concentrations or ratios of protein to nanodiscs.

This saves you time and effort because you don't have to spend months on working to optimize one nanodisc condition. You can screen, say, any protein discovery. You can screen up to eight different nanodiscs against your protein targets. Similarly, if you want to change different lipids depending on what your downstream needs are, you can screen a lot together. 

When I was working on structural biology, getting the structure sorted was a big bottleneck to plan into any downstream drug discovery workflow. If you have a structure available, it will be a big shift in paradigm because it will accelerate your target validation. You will have an early structural insight to confirm the druggability and to guide the rational design before any large-scale screening of your downstream ligands.

You can prioritize better, because once you have access to validated protein structures, you can triage your targets based on predicted binding pockets and you can look for allosteric sites. Once your structure-guided screening is about to start, it can facilitate the design of focused compound libraries. Because you already know what kind of binding pocket you're looking for, you can do molecular dynamics simulations on that to make your work faster. The best thing is, it will save a lot of time on hitting blind spots, because it avoids investment in undruggable targets by providing early functional structural feedback. 

In the current landscape, cell-free systems are ideal for producing toxic, difficult or modified proteins rapidly or flexibly. But for a lot of people, cell-based systems remain superior for post-translational modifications (PTMs) and for higher yield bulk production.

How I see the evolution of the two is using hybrid models, where we use cell-free systems for early screening and prototyping, and then we can switch to optimize cell-based production.

You save a lot of time if you already know what kind of media and expression conditions to go for and what kind of cell strains to pick. Depending on the constructs that you have tested in cell-free, you can get the best conditions picked and then think about your bulk cell-based scaleup.

Of course, there will be a reduction, because as cell-free systems become more affordable and customizable, they will move from the niche to mainstream, especially in early discovery and synthetic biology.

You can get tailored expression because the cell-free systems can evolve to accommodate a lot of things, even enzymes for doing PTMs. Even these issues can now be addressed with cell-free, so that's why I think a hybrid plan is good. 

If you think of conventional methods of protein biochemistry, you can imagine working with big bioreactors, centrifuges, hoods and incubators. If you're working in a cell-free system, energy will be saved because you don't have to maintain any bioreactors or cell cultures.

The waste is low because you are targeting expression of functional proteins, and it reduces the reagent consumption. You are not doing iterative trial and error cycles, so you're not using consumables – like plastics, reagents and media – on unsuccessful studies.

The production is localized because with cell-free systems the liquid handling is very miniaturized and it's a very portable system. You don't need a big footprint in the lab to place these cell-free units. It can be like a PCR machine in one corner, not taking up much space, and you can do so much in one go that it saves time doing multiple laboratory-based experiments for conventional biochemistry.

It's eco-friendly because of the replacement of detergents with biodegradable or less toxic materials. It also will minimize the cold chain logistics, such as the freezers and fridges that you need to maintain.


The introduction to this interview includes text that has been created with the assistance of generative AI and has undergone editorial review before publishing. Technology Networks' AI policy can be found here.