Membrane proteins act as gatekeepers, messengers, and engines for the cell. Embedded within membranes, they manage substance flow, receive external signals, and catalyze reactions. Their hydrophobic nature and association with the lipid bilayer make them difficult to isolate for study. Purifying them allows researchers to investigate their structure and function in detail.
Expression Systems for Membrane Proteins
The first step is producing the protein in large quantities using recombinant expression systems. The bacterium E. coli is a common choice for its rapid growth, but it often fails to correctly fold complex proteins or add molecular modifications. Researchers may instead use yeast systems, like Pichia pastoris, which can perform some modifications and generate high protein yields.
More complex proteins require expression systems that better mimic their native environment. Insect cells, used with a baculovirus vector, can produce large proteins with many post-translational modifications. For proteins demanding the most authentic processing, such as for human therapeutics, mammalian cell lines are preferred despite higher costs and lower yields, as they ensure correct folding and modifications.
Production can be improved through optimization strategies. The protein’s genetic code can be altered via codon optimization to match the expression host, enhancing synthesis rates. Small protein sequences, or tags, can also be fused to the membrane protein. These tags aid in the protein’s capture during purification and can improve its solubility and yield.
Solubilization of Membrane Proteins
Once produced, the target protein is extracted from the lipid membrane in a process called solubilization. The protein must be removed from its lipid environment without losing its native shape, which is tied to its function. The primary tools for this are detergents, which disrupt the lipid bilayer and shield the protein’s hydrophobic regions from the aqueous solution.
Detergents are categorized as non-ionic, ionic, and zwitterionic. Non-ionic detergents like n-dodecyl-β-D-maltoside (DDM) are mild and favored for preserving a protein’s structure and activity. A detergent must be used above its critical micelle concentration (CMC) to form micelles that encapsulate the protein, making it soluble. Choosing a detergent requires balancing solubilization efficiency with protein stability.
Newer solubilization agents that better mimic the native membrane environment are becoming more common. Amphipols are polymers that wrap around a membrane protein, keeping it soluble without detergents. Another approach uses styrene maleic acid (SMA) copolymers to excise a patch of the native membrane with the protein, creating a nanodisc. These methods provide a more stable environment, preserving the protein’s functional state for analysis.
Chromatographic Purification Strategies
After solubilization, the target protein exists in a mixture with other cellular components. A series of chromatographic techniques is used to isolate it from this mixture. These methods require solutions containing a detergent or other stabilizing agent to prevent the protein from aggregating.
Affinity chromatography is often the first step. This technique uses the specific tag engineered onto the protein, such as a His-tag or Strep-tag. The protein mixture is passed through a column with a resin that binds only to the tag, capturing the target protein while contaminants flow through. The bound protein is then released (eluted) by changing the buffer conditions, such as adding imidazole to release His-tagged proteins.
Further steps are often needed to achieve a pure sample. Ion-exchange chromatography (IEX) separates proteins based on their net surface charge. The final polishing step is often size-exclusion chromatography (SEC), which separates molecules by size. SEC is effective at removing remaining contaminants and aggregated forms of the target protein, yielding a highly pure sample.
Assessing Protein Purity and Quality
After purification, the protein’s purity, identity, and quality must be verified using several techniques.
- Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) separates proteins by size. A stained gel allows for visual assessment of purity, with a successful purification showing a single band at the expected molecular weight.
- Western blotting confirms the protein’s identity. After SDS-PAGE, proteins are transferred to a membrane and probed with an antibody that recognizes the target protein or its tag, confirming it is the correct one.
- Analytical size-exclusion chromatography assesses structural integrity. This technique determines if the protein is in a single state or has formed larger, non-functional aggregates.
- Spectroscopic methods like circular dichroism provide information about the protein’s secondary structure, helping to confirm that it has folded correctly and retained its native conformation.
Significance and Uses of Purified Membrane Proteins
Purifying membrane proteins enables many scientific investigations and applications. Purified, structurally intact proteins are required for determining their three-dimensional structures. Techniques like X-ray crystallography and cryo-electron microscopy (cryo-EM) use these samples to generate high-resolution atomic models, revealing how these molecular machines work.
Purification is also for understanding a protein’s function. With a pure sample, researchers can conduct functional assays, like measuring ion transport or how a receptor binds its ligand. This detailed characterization is not possible within the complex environment of a whole cell.
Membrane proteins are involved in many diseases, making them targets for drug development. Purified proteins are used in screening campaigns to identify drug candidates that can modulate their activity. They are also used to generate specific antibodies for use as therapeutics or as research tools.