Membrane Protein Production Protocol and FAQs

Membrane proteins are a class of important proteins that exist in the cell membrane, intima or plasma membrane. They play a key role in cell function, signal transduction, material transport, cell recognition, energy conversion and so on. Due to the special structure and function of membrane proteins, the research on their production and function has always been a research hotspot in many fields such as biology, pharmacology, structural biology and biotechnology.

Principle of Membrane Protein Production

The principle of membrane protein production involves several key steps, including membrane protein expression, folding, insertion into the membrane, and how to extract and purify from the membrane. The basic principle is as follows:

Selection of Expression Systems

Due to the hydrophobicity of membrane proteins, they are not easy to be stably expressed in an aqueous environment. Therefore, the selection of an appropriate expression system is essential for the production of membrane proteins. Commonly used expression systems include bacteria (such as E.coli), yeast, insect cells and mammalian cells. Each system has its advantages and disadvantages, and the choice of which system usually depends on the properties of membrane proteins and their applications.

Insertion and Folding of Membrane Proteins

Membrane proteins usually enter the membrane through endoplasmic reticulum or membrane protein synthesis channels. In this process, the ability of the protein to fold and insert into the membrane is crucial. Some membrane proteins require the assistance of specific cofactors or chaperone proteins to complete proper folding and function.

Extraction and Purification

Membrane proteins are usually stabilized in the cell membrane by interacting with the phospholipid bilayer. Extraction of membrane proteins requires the use of surfactants (such as Dodecyl β-D-maltoside, Triton X-100, etc.) to break the cell membrane and release membrane proteins. The role of these surfactants is to destroy the structure of the membrane while maintaining the function and structure of the membrane protein.

Dissolution and Complex Stability

The hydrophobic region of membrane protein makes it easy to aggregate or precipitate in water-soluble solutions, so appropriate dissolution buffers and auxiliary stabilizers are needed to maintain the stability of its structure.

The Steps of Membrane Protein Production

The production process of membrane proteins includes multiple steps : gene cloning, expression, extraction, purification, and functional detection.

Gene Cloning

The production of membrane proteins usually begins with the construction of expression vectors. First, the genes of the target membrane proteins need to be obtained. It can be obtained directly by PCR amplification or gene synthesis. The target gene is inserted into a suitable expression vector by molecular cloning technology, which usually contains alternative marker genes, such as antibiotic resistance genes. The target gene was cloned into the expression system by restriction enzyme cleavage, ligation and transformation.

Selection and Transformation of Expression System

Common membrane protein expression systems include :

1 E.coli system : By inserting the recombinant gene into the plasmid of E.coli, it is expressed in large quantities under induction conditions (such as IPTG induction). E.coli has the advantages of low cost and high production efficiency. However, due to the characteristics of membrane proteins, many membrane proteins cannot be correctly folded and inserted into the membrane in E.coli.
2 Yeast system : Yeast system can fold and insert into the membrane more correctly than E.coli, and is suitable for some membrane proteins that require glycosylation.
3 Insect cell system : Insect cells can better fold membrane proteins and obtain appropriate modifications by expressing viral vectors (such as Baculovirus). Insect cell systems are suitable for some of the more complex membrane proteins.
4 Mammalian cell system : Mammalian cell system can provide complete glycosylation and correct membrane protein folding, which is suitable for some membrane proteins with high glycosylation requirements or toxicity.

Culture and Induced Expression

After selecting the appropriate expression system, the transformed cells were grown in the appropriate medium. During the culture process, depending on the selected system, temperature regulation or inducer (such as IPTG) addition is usually required to promote the expression of membrane proteins. The expression of most membrane proteins needs to be carried out at low temperatures to avoid premature protein precipitation and misfolding.

Extraction and Dissolution of Membrane Proteins

Membrane proteins generally exist in the form of embedded membranes, which need to be released by breaking cells. There are many methods of cell disruption. Common methods include :

1 Ultrasonic disruption : The cell wall is broken by ultrasound to release membrane proteins.
2 High pressure homogenization : cell rupture by high pressure.
3 Chemical method : Use chemical reagents, such as Lysozyme, to dissolve cell membranes.

Then, appropriate surfactants (such as Dodecyl β-D-maltoside, Triton X-100, etc.) were added to help membrane proteins release from the membrane and maintain their solubility.

Purification of Membrane Protein

Purification of membrane proteins usually uses affinity chromatography, ion exchange chromatography, gel filtration chromatography and other techniques. Affinity chromatography usually uses tags (such as 6xHis tags) or antibody-specific purification of membrane proteins. Gel filtration is used to separate membrane proteins and other small molecular impurities according to molecular size. During the purification process, it is important to maintain the stability of membrane proteins. Stabilizers and low temperature conditions are often required to prevent degradation or aggregation of membrane proteins.

Functional Detection of Membrane Proteins

The purified membrane protein needs functional verification. Commonly used functional testing methods include :

1 Receptor binding assay : detect whether membrane proteins can bind to their target ligands or other molecules.
2 Determination of ion channel activity : If the membrane protein is an ion channel, the current change is detected by electrophysiological method.
3 Mass spectrometry analysis : further verify the structure and glycosylation of membrane proteins.

Figure 1. Basic considerations important for the initial design of CF expression processes. (Reference source: Membrane protein production in Escherichia coli cell-free lysates.）

Figure 2. Schematic comparison of cellular versus CF screening strategies for membrane protein production. (Reference source: Membrane protein production in Escherichia coli cell-free lysates.)

Application of Membrane Protein Production

Research on Drug Targets

Membrane proteins play an important role in cell signal transduction, receptor binding and transport, and thus become an important target for drug development. The study of membrane proteins is helpful to the development of new targeted drugs, especially in the fields of cancer, infection, cardiovascular disease and so on.

Vaccine Development

Some membrane proteins (such as surface proteins of pathogens) can be used as candidate antigens for vaccines. For example, the membrane protein of influenza virus is one of the main targets for the development of vaccines.

Structural Biology Research

The three-dimensional structure information of membrane proteins is critical for understanding their functions. Through X-ray crystallography, cryo-electron microscopy (Cryo-EM) and nuclear magnetic resonance (NMR) techniques, the relationship between the structure and function of membrane proteins can be further explored.

Biosensors

Membrane proteins, especially ion channels and receptor-like membrane proteins, can be used to develop biosensors. These sensors can be used for environmental monitoring, pathogen detection, etc.

Alpha Lifetech provides membrane protein production testing services designed to provide high-quality experimental solutions for life science research. Membrane proteins play a vital role in cell function, so it is particularly important to accurately analyze and produce them. We use advanced technology platform to realize the whole process service from small-scale laboratory preparation to large-scale production, and can customize the production plan according to customer needs.

FAQ

1. The expression of membrane proteins is usually much lower than that of soluble proteins.

Cause: This may be due to the hydrophobicity of the membrane protein, its insertion and folding mechanism in the cell membrane, and the requirements for the expression system, which makes its production efficiency low. Solutions: (1) Optimizing the expression system : Selecting the appropriate expression system is essential for improving the expression of membrane proteins. Common expression systems include Escherichia coli, yeast, insect cells and mammalian cells. For some proteins, yeast and insect cell systems may be more effective than E.coli. (2) Optimization of culture conditions : The expression conditions were optimized by adjusting the temperature, induction time and medium composition. For example, lower temperatures can slow down the misfolding and aggregation of membrane proteins in E.coli.
2. Membrane proteins usually need to be folded correctly to perform biological functions, but they may not fold correctly during expression, resulting in loss of function or aggregation precipitation.

Solutions : (1) The use of auxiliary means : chaperones were used to help membrane protein folding, or folding promoters, such as nonionic surfactants (such as Dodecyl β-D-maltoside, Triton X-100, etc.) were added to promote the correct folding of membrane proteins. (2) Low temperature culture : adjusting the temperature to slow down the expression of membrane proteins and reduce the misfolding and aggregation problems caused by overexpression. (3) Using different membrane protein fusion proteins : fusion proteins (such as green fluorescent protein (GFP) or affinity tags) that are suitable for folding can be added to the N-terminus or C-terminus of the membrane protein to help the membrane protein fold correctly.
3. Membrane proteins may be unstable during extraction and purification, easy to degrade or aggregate, resulting in low yield and difficulty in obtaining a sufficient number of pure products.

Solutions : (1) The use of stabilizers : Stabilizers (such as glycerol, glucose, Ca2 +, etc.) were added during the extraction and purification of membrane proteins to help maintain their structural stability. (2) The use of surfactants : The use of appropriate concentrations of surfactants (such as Dodecyl β-D-maltoside, Triton X-100) can help membrane proteins remain stable in solution and prevent their aggregation. (3) Cryopreservation : Membrane proteins should be preserved at low temperatures to avoid degradation or loss of activity at high temperatures. The purified membrane protein can be frozen at − 80 ° C and avoid repeated freezing and thawing.
4. Membrane proteins are embedded in the cell membrane and are usually not easily extracted from the cell membrane. Even using common membrane breaking methods, the extraction of membrane proteins may still be inefficient.

Solutions : (1) Use of suitable surfactants : The extraction of membrane proteins usually relies on surfactants to destroy cell membranes and dissolve membrane proteins. The commonly used surfactants include Triton X-100, Dodecyl maltoside, n-dodecyl-β-D-maltoside and so on. When selecting surfactants, it is necessary to balance their ability to effectively extract membrane proteins while avoiding damage to the structure and function of proteins. (2) Ultrasonication or high pressure homogenization : The cell wall was broken by physical methods (such as ultrasonication or high pressure homogenization) to extract intracellular membrane proteins. (3) Optimization of membrane breaking conditions : adjust the concentration of surfactant, membrane breaking time and temperature, etc., to ensure that the membrane protein can be effectively released into the solution.

reference

[1] Schavemaker PE, Poolman B. (Membrane) Protein Production in Context. Trends Biochem Sci. 2018;43(11):858-868. doi:10.1016/j.tibs.2018.08.009

[2] Vaitsopoulou A, Depping P, Bill RM, Goddard AD, Rothnie AJ. Membrane Protein Production in Insect Cells. Methods Mol Biol. 2022;2507:223-240. doi:10.1007/978-1-0716-2368-8_12

[3] McIlwain BC, Kermani AA. Membrane Protein Production in Escherichia coli. Methods Mol Biol. 2020;2127:13-27. doi:10.1007/978-1-0716-0373-4_2

[4] Henrich E, Hein C, Dötsch V, Bernhard F. Membrane protein production in Escherichia coli cell-free lysates. FEBS Lett. 2015;589(15):1713-1722. doi:10.1016/j.febslet.2015.04.045