Membrane Protein Production in Nanodiscs
Membrane proteins (MPs) are essential to many organisms’ major functions. They are notorious for being difficult to isolate and study, and mimicking native conditions for studies in vitro has proved to be a challenge. Lipid nanodiscs are among the most promising platforms for MP reconstitution, but they contain a relatively labile lipid bilayer and their use requires previous protein solubilization in detergent. These limitations have led to the testing of copolymers in new types of nanodisc platforms. Polymer-encased nanodiscs and polymer nanodiscs support functional MPs and address some of the limitations present in other MP reconstitution platforms. To purify membrane protein in a native-like form and a condition without detergent, Beta Lifescience developed the most up-to-date platform.
Nanodiscs refer to small (7-50 nm in diameter) disk-like phospholipid bilayers consisting of two components: phospholipids, which can be synthetic phospholipids or native cell membrane phospholipids and stable belts that closely surround the phospholipids, including membrane scaffold proteins (MSPs) or synthetic polymers(SMA).
As mentioned before nanodiscs can be differentiated between their phospholipid composition and most importantly, their type of stabilizer. This stabilizer is the reason why nanodiscs in total are split into two main categories: MSP nanodiscs and Synthetic nanodiscs.
Composition and Structure of Nanodisc
Two Types of Nanodisc
As mentioned before nanodiscs can be differentiated between their phospholipid composition and most importantly, their type of stabilizer. This stabilizer is the reason why nanodiscs in total are split into two main categories: MSP nanodiscs and synthetic nanodisc. The respective names originate from the type of stabilizer that is used to keep the nanodiscs together and form them in the first place. It also decides what the lipid composition of the nanodisc is made up of. MSP nanodiscs always contain an artificial lipid composition. Meaning you have full control of it. In contrast: SMA uses the native cell phospholipids to create the nanodisc.
Figure 1. Two types of nanodiscs: synthetic nanodiscs (blue stabilizer) and MSP nanodiscs (green stabilizer)
![](http://www.betalifesci.com/cdn/shop/files/unnamed_07dacd73-c6ef-40fa-983e-ca8b41c0ae12_{width}x.png?v=1721339825)
SMA Nanodiscs
The assembly of a synthetic nanodisc begins with a complete cell. It uses synthetic polymers to dissolve cell membranes while using native cell phospholipids to form nanodisc structures around membrane proteins. The polymer acts as both a solvent enhancer and stabilizer. Therefore, no additional detergent is required. Compared with the MSP nanodisc, synthetic nanodisc omits the screening process of detergent and phospholipid, thus shortening the development cycle. Synthetic nanodisc uses synthetic polymers that are non-protein components and therefore have a higher purity than MSP nanodiscs.
MSP Nanodiscs
MSPs closely wrap around the artificial phospholipids, resulting in the formation of MSP nanodisc. MSPs are truncated forms of apolipoprotein A-I (ApoA-I) and act as stabilizers. Each MSP protein contains 200 amino acid residues and is soft and malleable. MSP provides a hydrophobic surface facing the hydrophobic tail of the lipids, and a hydrophilic surface on the outside. This setup makes nanodiscs highly soluble in aqueous solutions. Once assembled into nanodiscs, membrane proteins can be kept in solution without detergents.
Among MSPs, MSP1D1 and MSP1EE3D1 are the two most widely used granules. The nanodiscs prepared by this method can maximize the preservation of the natural conformation of membrane proteins during purification for further study. The diameter of nanodiscs is determined by the length of the MSP band and when the phospholipid is in the optimum molar ratio to MSP, uniform nanodiscs are formed.
- Assembly of Membrane Proteins Dissolved in the Detergent: The membrane proteins are first purified in the presence of detergents and then spontaneously assembled into nanodiscs with the added MSPs and phospholipids. After removing the detergents by dialysis or hydrophobic adsorbents, the forming nanodiscs can be purified by dimensional exclusion chromatography or affinity purification.
- Nanodisc Combination with and Cell-free Expression System: The assembled nanodiscs are added to the cell-free expression system. Multiple nanodiscs are obtained by introducing MSP particles into the mixture containing membrane proteins and adding excess lipids. The mixture of nanodiscs is purified to obtain nanodiscs only expressing the target membrane proteins. This method does not require the addition of detergents, which can minimize the presence of artifacts.
![Table 1: Small comparison between MSP and SMA](http://www.betalifesci.com/cdn/shop/files/unnamed_c47913a2-1030-4c4e-af7e-dd6154ff9ba0_{width}x.png?v=1721340308)
![Figure 2. (a) SMA-Nanodisc assembly process diagram; (b)MSP-Nanodisc assembly process diagram](http://www.betalifesci.com/cdn/shop/files/unnamed_6bebb274-2cbc-403e-891f-5041915eef9f_{width}x.png?v=1721340423)
Nanodisc Platform
A widely used approach to isolate membrane proteins involves using detergents to dissolve the lipid bilayer matrix. This process typically results in the creation of tiny spherical structures, called micelles that contain the membrane protein of interest, detergent molecules, and any remaining lipids. Two types of Nanodisc platforms can be used to extract different membrane proteins with varying properties from membranes. In the styrene–maleic anhydride copolymer (SMA) assembly-based SMA–Nanodisc platform (a), SMA binds to the surface of the membrane and then inserts into its hydrophobic core. Finally, the membrane is solubilized, and Nanodiscs are formed. In the membrane skeletal protein (MSP)-based MSP–Nanodisc platform (b), the membrane proteins are first prepared using a detergent and then assembled via the addition of phospholipid molecules and MSP. By adjusting the ratio of phospholipids, MSP, and membrane proteins, the membrane proteins to be assembled can be clustered in different states on Nanodiscs.
Beta Lifescience has successfully built the Nanodisc platform, and SMA copolymers are highly promising, versatile tools for the study of diverse membrane-related processes that will play a significant role in advancing membrane research in the future.
Application of Nanodisc in Membrane Proteins
Nanodiscs are widely used in the study of membrane proteins due to their ability to provide a stable, native-like environment. Here are some key applications of nanodiscs in membrane protein research:
Structural Biology
Cryo-Electron Microscopy (Cryo-EM):
Nanodiscs provide a suitable environment for high-resolution structural studies of membrane proteins using Cryo-EM, allowing researchers to visualize the protein in near-native conditions.
X-ray Crystallography:
Nanodiscs can be used to form crystals of membrane proteins, facilitating X-ray crystallography studies.
Biochemical and Biophysical Characterization:
- Functional Assays: Nanodiscs maintain the functional activity of membrane proteins, enabling various functional assays such as ligand binding, enzyme activity, and ion transport studies.
- Spectroscopy: Techniques like nuclear magnetic resonance (NMR), fluorescence spectroscopy, and electron paramagnetic resonance (EPR) benefit from the stable, native-like environment provided by nanodiscs.
Drug Discovery and Screening: High-Throughput Screening
Nanodiscs can be used in high-throughput screening assays to identify potential drug candidates targeting membrane proteins. Drug Binding Studies: Nanodiscs facilitate detailed studies of drug binding and interaction with membrane proteins, providing insights into the mechanism of action and binding kinetics.
Reconstitution of Membrane Protein Complexes
Protein-Protein Interactions: Nanodiscs enable the study of interactions between membrane proteins and other biomolecules, such as signaling partners, chaperones, and other membrane proteins. Complex Assembly: Researchers can reconstitute multi-protein complexes in nanodiscs to study their assembly and function.
Immune Response and Vaccine Development
Antigen Presentation: Membrane proteins reconstituted in nanodiscs can be used as antigens in vaccine development, enhancing the immune response by presenting the protein in a more native-like state. Adjuvant Studies: Nanodiscs can serve as adjuvants to improve the efficacy of vaccines by stabilizing and presenting membrane protein antigens effectively.
Overall, nanodiscs are versatile tools that enable detailed and accurate studies of membrane proteins, contributing to advancements in structural biology, drug discovery, vaccine development, and more.
Advantages of Membrane Protein Expression in Nanodisc platform
- High biological activity: The Nanodisc platform provides an environment similar to the cell membrane, showcasing the complete conformation and preserving biological activity.
- High stability and compatibility: The Nanodisc platform is composed of high-quality lipids and proteins, exhibiting excellent stability and biological compatibility, and providing reliable experimental results.
- Advantages in membrane protein expression: The Nanodisc technology platform offered by Beta Lifescience exhibits outstanding advantages in membrane protein expression. By providing an environment similar to the cell membrane, Nanodiscs can stably encapsulate and express various types of membrane proteins, including G protein-coupled receptors, ion channel proteins, and other challenging-to-express membrane proteins.**
FAQs
The effect of non-specific antibodies should not be ignored, the use of membrane proteins under the VLP and Nanodisc platforms may indeed generate non-specific antibodies, both platforms have corresponding blanks for counter-screening to remove non-specific antibodies: VLP-membrane proteins have an isotype control product, VLP blank; Nanodisc has two MSP1D1 blanks, and one of the MSP1D1 can be used as an isotype control for the tag free membrane proteins, and the other one can be used as an isotype control if biotinylated Nanodisc-membrane proteins are used. tag free membrane proteins, and if biotinylated Nanodisc-membrane proteins are used, the other MSP1D1 can be used as an isotype control.
Based on our experiences, proteins can exhibit bands at higher molecular weights due to post-translational modifications (PTMs) such as phosphorylation and glycosylation, or bands at lower molecular weights due to cleavage during cellular processes or sample preparation. Therefore, we suggest checking the literature to see if multiple bands are reported for the specific protein.
We customize Nanodisc development of new target proteins. We typically use expression cDNA clones for test runs, and proteins obtained from the test runs, ranging from ng to 100ug, are sent to the customer. The actual cost and time depends on the specific project requirements. For example, a protein with 12 transmembrane structural domains will incur a higher cost in testing compared to a protein with 4 transmembrane structural domains. In addition, any other considerations for a customized project, such as unique specifications or special handling instructions, can be discussed based on project details. If the customer does not provide an expression cDNA clone, we will charge separately, in which case we will send the DNA clone to the customer.
VLP depends on the target, there are some targets VLP expression is not very good, the normal expression of almost in 1mg or so; Nanodisc yield is lower, probably in the μg level, so relatively high cost, but the protein purity is also relatively high, is the purification of the target protein with natural conformation.
Custom Services
We have rich experience in the expression of recombinant proteins (antibodies, enzymes, membrane proteins, cytokines, etc.) in any species. We provide recombinant protein expression and purification services from small-scale to large-scale.
Beta Lifescience has accumulated considerable experience and expertise in protein crystallization service. We will provide you the one-stop solution according to our customer's request.
Beta Lifescience had developed our unique antibody production platform for high-quality and comprehensive custom antibody production services covering monoclonal and polyclonal development.
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