Tackling Insoluble and Difficult-to-Express Proteins

Few things in the lab can feel as challenging as working with transmembrane proteins. These complex structures span cell membranes, performing vital functions from signal transduction to transport of molecules across cellular barriers.

Low expression yields, protein misfolding, and aggregation frequently plague researchers attempting to produce functional transmembrane proteins. Extracting these proteins from their native membrane environment without compromising structure and function requires specialized techniques and careful optimization—challenges that can derail even well-planned experiments.

Fortunately, advances in expression systems have created new opportunities for high-yield transmembrane protein production. This guide explores proven strategies to tackle common expression challenges, from selecting the right expression system to optimizing solubilization and purification methods.

Table of contents

• What is a transmembrane protein?
  • Integral vs peripheral membrane proteins
  • Structural features and hydrophobic chemistry of transmembrane proteins
  • Functional roles of transmembrane proteins in cell biology
• The importance of proteins in research discovery
• Key types of transmembrane proteins in research
  • Single-pass (bitopic) vs multi-pass (polytopic) proteins
  • GPCRs
  • Ion channels and carrier proteins
  • Diffusion mechanism proteins
• Transmembrane protein expression
  • Why are transmembrane proteins so difficult to express?
  • Common transmembrane protein expression systems
• HEK293 mammalian expression systems
  • Glycosylation and protein folding considerations
• Tips for improving transmembrane protein expression in mammalian systems
• More transmembrane protein resources

What is a transmembrane protein?

Transmembrane proteins are crucial for various cellular functions, including signaling, transporting, and maintaining structural integrity.

Integral vs peripheral membrane proteins

A transmembrane proteinis foremost a type of integral membrane protein (IMP). IMP proteins span the entire lipid bilayer, positioning parts on both the extracellular and cytoplasmic sides of the membrane.

This anchored positioning within the protective, hydrophobic core of the cell membrane bilayer has implications for research analysis. IMP and transmembrane proteins can only be extracted using detergents or other disruptive methods that disturb the membrane’s integrity.

Peripheral membrane proteins, on the other hand, are not embedded within the lipid bilayer. Instead, they are loosely attached to the membrane’s surface or to integral proteins through non-covalent interactions like hydrogen bonds and electrostatic forces. Due to their external association, they can be removed without disrupting the membrane, typically by altering pH or ionic strength.

Structural features and hydrophobic chemistry of transmembrane proteins

Because much of the challenge of studying transmembrane proteins comes down to molecular polarity, understanding the basic chemistry of these proteins is key.

1. Hydrophobic transmembrane domains: The segments of transmembrane proteins that traverse the lipid bilayer are typically composed of hydrophobic amino acids, allowing stable integration within the hydrophobic core of the membrane. These regions often form α-helices or β-barrels, depending on the protein’s specific structure and function.
2. Hydrophilic extracellular and cytoplasmic domains: The portions of the protein that extend beyond the membrane interact with the aqueous environments inside and outside the cell. These regions are rich in hydrophilic amino acids, facilitating interactions with other molecules and cellular components.
3. Amphipathic nature: Transmembrane proteins exhibit amphipathic characteristics, with both hydrophobic and hydrophilic regions, enabling them to interact with the diverse environments presented by the lipid bilayer and the aqueous cellular compartments.

Functional roles of transmembrane proteins in cell biology

Transmembrane proteins serve many critical biological functions, including:

• Transport: Facilitating the movement of ions and molecules across the membrane.
• Signal transduction: Acting as receptors for signaling molecules, initiating cellular and immune responses.
• Cell-cell interactions: Mediating interactions between cells and the extracellular matrix.
• Enzymatic activity: Catalyzing specific biochemical reactions at the membrane interface.
• Energy production: Facilitating mitochondrial and chloroplast energy creation processes like oxidative phosphorylation and photophosphorylation.
• Cell structure: Anchoring the cytoskeleton to the membrane, maintaining cell polarity, and driving morphogenesis.

The importance of transmembrane proteins in research discovery

The importance of transmembrane proteins to life science discovery cannot be overstated. Malfunctions in membrane proteins are implicated in a vast array of diseases – everything from chronic conditions like diabetes and heart disease to acute cases like cancer.

For this reason, more than half of all pharmaceuticals today target membrane proteins.

Key types of transmembrane proteins in research

There are many types of transmembrane proteins, and they are classified based on their structural organization, function, and number of times they span the membrane.

Single-pass (bitopic) vs multi-pass (polytopic) proteins

In transmembrane protein biology, it matters how many times the protein molecule intersects the cell membrane lipid bilayer.

Single-pass (bitopic) proteins traverse the lipid bilayer just once. They typically feature an extracellular domain for ligand binding and an intracellular domain for signaling. This relatively simple topology makes them more accessible for antibody development and structural studies.

Multi-pass (polytopic) proteins span the membrane multiple times, forming complex structures like channels or transporters. Their intricate architecture poses more challenges for structural characterization and therapeutic targeting.

G-protein coupled receptors (GPCRs)

GPCRs represent a major class of drug targets, and nearly all drugs targeting membrane proteins involve GPCR receptor targets.

GPCRs are characterized by seven transmembrane α-helices. They transduce extracellular signals, such as hormones and neurotransmitters, into intracellular responses via G protein activation.

Ion channels and carrier proteins

Ion channels are multi-pass proteins that form pores allowing selective passage of ions across membranes – crucial for processes like nerve impulse transmission and muscle contraction.

Carrier proteins like transporters and permeases facilitate the movement of molecules like glucose and amino acids across membranes, often undergoing conformational changes to transport their substrates.

Nuclear pore complexes (NPCs) represent one emerging area of research in this group; these large assemblies regulate the transport of macromolecules between the nucleus and cytoplasm but may also play an important role in genome stability and gene expression regulation.

Transmembrane protein expression

When expressing transmembrane proteins, selecting the appropriate system is crucial.

Why are transmembrane proteins so difficult to express?

While membrane proteins account for nearly 30% of all known proteins, they represent only 2-5% of the 3D structural protein data entries in core global repositories like the Protein Data Bank.

Why is that? In the simplest terms, working with transmembrane proteins is hard. Expressing transmembrane proteins at the bench is an art in itself that researchers are constantly endeavoring to improve and simplify.

For example: the hydrophobic regions of these proteins which interact with the lipid bilayer tend to aggregate when expressed in aqueous environment. This aggregation can lead to misfolding and loss of function, complicating study.

Another challenge is the low natural abundance of many membrane proteins; boosting expression levels usable levels for research can trigger host toxicity as a secondary obstacle.

A complex landscape of post-translational modification and the loss of the natural, protective lipid membrane environment needed for proper transmembrane protein folding and function adds another element of complication to expression and purification workflows.

There are strategies to approach these many obstacles, however, and move therapeutic development work forward.

Common transmembrane protein expression systems

Researchers commonly use four types of protein expression systems.

• Prokaryotic – Prokaryotic expression systems like E. coli or yeast are popular choices for early trials. Prokaryotic models offer fast growth rate and simpler genetic manipulation. Most lack the ability for post-translational modification, however.
• Insect – Insect cell systems, which often use baculovirus vectors, can be effective for expressing complex eukaryotic proteins. Glycosylation patterns may differ from those of mammalian cells, though.
• Mammalian – Mammalian expression systems, such as HEK293 or CHO cells, are ideal for producing properly folded proteins requiring human-like post-translational modifications.
• Cell-free – Cell-free expression systems synthesize proteins in vitro without living cells, using extracts containing the necessary transcription and translation machinery. These systems can allow rapid protein production and be especially suitable for toxic or unstable proteins. Post-translational modification capabilities may be limited.

HEK293 mammalian expression systems

The Gibco Expi293F expression system has become a strong choice for mammalian transmembrane protein expression, especially for G-protein coupled receptors (GPCRs). This human embryonic kidney cell line offers several compelling advantages:

• Rapid growth in suspension culture
• High transfection efficiency
• Capability to perform complex post-translational modifications
• Mammalian folding machinery appropriate for human proteins

A specialized variant—Expi293F GnTI- cells—provides significant benefits for structural biology applications. These cells lack N-acetylglucosaminyltransferase I (GnTI), an enzyme essential for complex N-glycan formation.

Glycosylation and protein folding considerations

The glycosylation profile of your expressed transmembrane protein can significantly impact its structure, function, and stability—making this a critical factor when selecting an expression system.

Expi293F cells produce human-like glycosylation patterns with complex, heterogeneous N-glycans across several species of transmembrane proteins. This heterogeneity, while representative of natural human proteins, can complicate structural studies by introducing sample variability. Expi293F GnTI- cells generate simpler, more homogeneous glycans.

Consider these essential factors related to your target proteins when choosing between expression systems:

• Research purpose: Structural studies benefit from the homogeneous glycosylation of Expi293F GnTI- cells, while functional studies may prefer standard Expi293F for more native-like modifications.
• Scale requirements: Both Expi293 lines are easily scaled in shake flasks typically delivers higher yields at large scale.
• Complexity of the target protein: Highly complex human transmembrane proteins with multiple glycosylation sites and palmytoilation sites may perform better in human-derived Expi293F cells.
• Downstream applications: Proteins destined for crystallization or cryo-EM benefit from the glycan homogeneity of GnTI- systems.

CTA Callout: Use the Gibco protein expression system tool to find your appropriate protein expression product lineup. Link: https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-expression/protein-expression-host-systems.html

Tips for improving transmembrane protein expression in mammalian systems

In general, the more difficult a protein is to express, the more important it is to optimize the expression. These optimizations can be lengthy and complex. Researchers should consider the end goal of the protein production run and what the downstream use of the purified protein will be. High yield does not always mean high activity

If possible, consider using a fusion of the protein of interest with an easy to quantify reporter, like GFP, to ease the quantification while optimizing expression using a DoE approach. These model proteins should be carefully validated. 

Some parameters to take into consideration to optimize from a protein expression system perspective:

• Design of DNA sequence. Leave nature to nature or optimize codonsWhile codon optimization may lead to higher levels of protein expression, pushing too much transcript too fast through the protein synthesis machinery may lead to loss in activity.
• Amount of transfected DNA. This will allow finetuning of expression level
• Time of expression duration. While longer expression runs may yield more protein, activity may be compromised. In general, cells should be harvested when cell viability goes below 80%. Dying cells don’t make pretty proteins.

Some membrane proteins may be more stable in the presence of a ligand, agonist or antagonist. This may be added to the cell culture during expression and/or harvest.

For further reading on how we optimized expression of a GPCR in Expi293, we recommend viewing our publication, “Thermostability of a recombinant G protein-coupled receptor expressed at a high level in mammalian cell culture” (Nature Research Scientific Reports 2020).

Conclusion

Transmembrane proteins are difficult subjects to tame, but the stakes for therapeutic potential are high. Every successfully expressed transmembrane protein feels like a small victory.

And the future remains bright, with membrane proteins being solved faster than ever before thanks to the combination of optimized protein production solutions and state-of-the-art structural analytical tools.

Interested in learning more? Explore the extended resource library below.

More transmembrane protein reading & resources

• Publication: Thermostability of a recombinant G protein-coupled receptor expressed at a high level in mammalian cell culture (Nature Research Scientific Reports 2020)
• Publication: Efficient and economic protein labeling for NMR in mammalian expression systems: application to a preT-cell and T-cell receptor protein (Protein Science 2024)
• Publication: Probing the conformational space of the cannabinoid receptor 2 and a systematic investigation of DNP-enhanced MAS NMR spectroscopy of proteins in detergent micelles (ACS Omega 2023)
• Poster: Strategies for high-titer protein expression using the ExpiCHO and Expi293 Transient Expression Systems
• Poster: Optimized expression of membrane proteins in the Expi293 and ExpiCHO Expression systems