DIMA Biotechnology LTD

Synthetic Nanodisc, A Promising Solution for“Undruggable” Membrane Proteins
Membrane proteins (MPs) play a critical role in various biological processes, such as facilitating materialtransport, signal transduction and enabling cell-to-cell recognition. They are frequently targeted bycurrently marketed therapeutics. In fact, over 60% of known drug targets specifically aim at membraneproteins such as BCMA, GPRC5D, CCR8 [1]. These proteins have been identified as key players innumerous diseases, including cancer, cardiovascular disorders, neurological conditions, and autoimmune
disorders. By targeting MPs, these drugs aim to modulate their activity and restore normal cellular function,offering potential treatments for a wide range of medical conditions.
Despite the crucial roles of MPs, they have been proven to be challenging to work with, especiallymulti-pass transmembrane proteins, which refer to proteins with two or more hydrophobic transmembraneregions. Because of these hydrophobic transmembrane regions, multi-pass transmembrane proteins are easy
to aggregate in vitro, making the isolation and purification of these proteins a technically demanding task.
Additionally, their three-dimensional structure is heavily influenced by surrounding environment, making it
difficult to maintain protein conformation during purification, which further complicate their study andanalysis.
Instead of using extracellular domains (ECDs) as what we normally do for single-pass MPs, multi-passtransmembrane proteins require full-length proteins in order to develop functional drugs against these
proteins. DIMA’s unique DiMProTM Synthetic Nanodisc technology provides a promising solution for these“undruggable” targets.

1. What is nanodisc protein?
2. What are the advantages of nanodisc platform for MPs?
3. What are the types of current nanodisc proteins?
4. Which type of nanodisc should you use for your proteins?
5. Why do we use mammalian cell expression system to produce synthetic nanodisc proteins?
6. Featured synthetic nanodisc products from DIMA Biotech
7. Featured Data from DIMA’s Synthetic Nanodisc Proteins
Wuhan institute of Biotechnology B7, Biolake No.666 Gaoxin Road, Wuhan, Hubei, China 430074+86-400-006-0995
info@dimabio.com
www.dimabio.com
1DIMA Biotechnology LTD

1. What is nanodisc protein?
The concept of nanodisc was first proposed by Professor Stephen G. Sligar from University of Illinois in
2002. In the late 1990s, Professor Sligar came across the groundbreaking research conducted by Professor
Ana Jonas at the same university while searching for good candidates for atomic force microscopes (AFM)
structural studies. Professor Jonas’s studies focused on the predominant circulating forms of high-density
lipoproteins (HDL) in the atherosclerotic process. She found that these forms typically exist as balls ofvariable size containing cholesterol esters, lipids, and proteins. However, she also discovered a transient
form of HDL that is roughly discoidal in shape and is stabilized by the amphipathic apolipoprotein A-I(Apo-AI). Inspired by Professor Jonas's work, Professor Sligar proposed the concept of Nanodiscs as a
powerful tool for studying the structure and function of membrane proteins.
The team reconstructed larger HDL particles by removing detergents from a lipid-enriched Apo-AI fraction
isolated from human blood and imaged these HDL particles on atomically flat mica [2] [3]. They found thatthese particles were extremely heterogeneous, with a peak in the size distribution corresponding to thelength of the repeating Apo-AI protein, which indicated there was a specific size range in which theseparticles were commonly found [4]. To create uniform-sized nanoparticles, Professor Sligar synthesizedrecombinant Apo-AI coding sequence that could be expressed in in E. coli and modified the remodelingprocedure. As a result of this genetic engineering and remodeling procedure, the researchers successfully
produced a group of proteins called "membrane scaffold proteins" (MSPs) which can self-assemble into
disc-shaped phospholipid bilayers known as Nanodiscs. These Nanodiscs were wrapped with anamphipathic helical band surrounding the phospholipid alkyl chains that can mimic the natural environmentof membrane proteins and provide a stable platform for their analysis [5]. Since its introduction, Nanodiscs
have revolutionized the field of membrane protein studies and become a valuable tool for drug discovery
and delivery as well. Their small size, stability, and ability to mimic cell membranes make them ideal
carriers for delivering therapeutic.
Nanodisc proteins are nanoscale structures composed of phospholipid bilayer and MPs. They are typically
disc-shaped multi-pass transmembrane proteins with a diameter of around 10-20 nanometers. Nanodisc
proteins use stabilizers, such as MSP, to provide a stable environment for incorporating the MPs. The
hydrophobic face of the stabilizer interacts with the inner lipid layer, facilitating the integration of MPs into
Wuhan institute of Biotechnology B7, Biolake No.666 Gaoxin Road, Wuhan, Hubei, China 430074+86-400-006-0995
info@dimabio.com
www.dimabio.com
2DIMA Biotechnology LTD
Nanodisc, which preserves the natural conformation and activity of MPs. The hydrophilic side ofNanodiscs provides high solubility and stability in aqueous solution, further enhancing their usefulness as aresearch tool.
Nanodiscs provide a very stable environment for MPs in vitro, making them ideal for studying ligand
binding or investigating the effects of antagonists and agonists using NMR and SPR. Nanodisc proteins canalso be used in various other fields, including Resonance Raman, Cryo-electron microscopy (Cryo-EM),
Matrix-Assisted Laser Desorption Ionization (MALDI), protein activity studies, and time-resolved
fluorescence spectroscopy. Furthermore, the assembly of antigens into Nanodiscs has shown promise inenhancing the immune response of mice, demonstrating their great potential for use in vaccine preparation
and antibody production.

2. What are advantages of Nanodisc platform for MPs?
As mentioned before, membrane proteins account for more than 60% of all FDA-approved drug targets and90% of antibody-based drug targets. To prepare functional multi-pass transmembrane protein, various
cellular and cell-free expression systems have been employed. Besides Nanodisc, there are severalsolutions for multi-pass transmembrane protein preparation, including detergent, peptide made up ofN-terminus or extracellular loops, membrane fraction, whole cell and VLP [6].
Detergent is widely used to produce multi-pass transmembrane proteins. However, one of the biggest
downsides of using detergent is proteins need to be maintained in detergent-containing solutions, anddetergent cannot be completely removed. This can be problematic for downstream applications, as thepresence of detergent may interfere with certain experiments or affect the protein structure.
An alternative method to obtain proteins with good solubility and correct folding is to use peptides derived
from the N-terminus or extracellular loops. However, the products may not resemble the structural featuresand conformation of the original extracellular loops of multi-pass transmembrane proteins in the nativemembrane.
Another option is using membrane fractions, whole cells or VLPs to maintain natural structure ofmulti-pass transmembrane proteins, but immunization with these products will result in high levels ofnonspecific antibody background.
Comparing with these traditional solutions, Nanodisc platform offers a more advanced solution. It enables
Wuhan institute of Biotechnology B7, Biolake No.666 Gaoxin Road, Wuhan, Hubei, China 430074+86-400-006-0995
info@dimabio.com
www.dimabio.com
3

DIMA Biotechnology LTD
the production of full-length MPs with high purity and excellent solubility in aqueous solutions. Proteins
incorporated into Nanodiscs are in native membrane environment, ensuring they remain biologically active.
There is no detergent in these products, making them suitable to use in cell- based assays and otherexperiments.

3. What are the current Nanodisc protein types?
Nanodisc proteins have gained significant interest in recent years due to their ability to mimic the natural
lipid environment and provide a stable platform for membrane proteins. There are two types of Nanodisc
proteins according to different stabilizers: Membrane scaffold protein (MSP) Nanodisc and Synthetic
Nanodisc proteins.

Figure 1. The structure of MSP-Nanodisc (left) and Synthetic Nanodisc (right)

3.1 MSP nanodisc
MSP nanodisc uses a specific protein called Membrane Scaffold Protein (MSP) as a stabilizer to wrap
artificial phospholipid components and transmembrane proteins to form a nanodisc. This type of nanodisc
is commonly used for membrane protein production before polymer Nanodisc and has been extensively
studied.
This type of Nanodisc protein consists of a hydrophobic surface towards the inner lipid layer and ahydrophilic surface towards the outer layer, which allows MPs to be highly soluble in aqueous solutions
even in the absence of detergents. The size of MSP Nanodisc protein is determined by the selected MSPs
which usually have a diameter ranging from 7 to 13 nanometers in diameter. There are several commonlyused MSPs in Nanodisc assembly, including MSP1D1, MSP1D1-DH5, and MSP1E3D1. These MSPs havebeen extensively studied and proven to be effective in generating stable and functional Nanodisc proteins.
The preparation of MSP Nanodisc proteins can be achieved through the following two methods.
Assembly of Membrane Proteins Dissolved in Detergent
Wuhan institute of Biotechnology B7, Biolake No.666 Gaoxin Road, Wuhan, Hubei, China 430074+86-400-006-0995
info@dimabio.com
www.dimabio.com
4DIMA Biotechnology LTD
The first method involves the assembly of membrane proteins that have been dissolved in detergents. Afterthe purification of membrane proteins in the presence of detergents, MSPs and phospholipids are added tothe solution. The combination of membrane proteins, MSPs, and phospholipids leads to the spontaneous
formation of Nanodiscs. To obtain purified Nanodisc proteins, detergents are removed through size
exclusion chromatography or affinity purification. The figure below takes the A2AR protein as an example
to illustrate the specific process.

Figure 2. Assembly of MSP-Nanodisc [7]Combination of Nanodisc and Cell-free expression System

Alternatively, Nanodiscs can be generated by combining them with a cell-free expression system. In thisapproach, pre-assembled Nanodiscs are added to a cell-free reaction system. Starting from expression
plasmids, membrane proteins are produced in a cell-free system and then integrated spontaneously intoNanodiscs. This method provides a convenient way to produce Nanodiscs with specific MPs. Theirhydrophobic-hydrophilic arrangement, along with the flexibility of the assembly process, makes themvaluable tools in various fields of biological research.

3.2 Synthetic Nanodisc
Unlike MSP Nanodisc, synthetic Nanodisc is produced with synthetic polymers, which usually include
styrene-maleic acid co-polymers (SMAs) and Diisobutylene-maleic acid (DIBMA). Each polymer has its
own advantages and disadvantages. For example, DIBMA doesn’t absorb light at 280 nm, which makes it
an ideal choice for experiments that involve protein analysis using spectroscopy techniques. The assembly
Wuhan institute of Biotechnology B7, Biolake No.666 Gaoxin Road, Wuhan, Hubei, China 430074+86-400-006-0995
info@dimabio.com
www.dimabio.com
5DIMA Biotechnology LTDof synthetic Nanodisc protein starts with intact cells. The long chains of synthesized polymers can beinserted into the cell membrane surrounding the desired MPs. As a result, the MPs detach from themembrane and dissolve in the polymer solution. The interaction between polymer and MPs leads to thestabilization of MPs in the newly formed Nanodisc protein (As shown in Figure 4). Overall, the use ofsynthetic polymers in the production of Nanodiscs offers researchers more versatility and control over thestabilization and analysis of MPs. This method allows for the creation of stable and functional Nanodisc
proteins that can be utilized in a wide range of biochemical and biophysical studies.

Figure 3. Assembly of Synthetic Nanodisc

4. Which type of nanodisc should you use for your proteins?
Both MSP and synthetic Nanodisc proteins are designed to achieve solubilization and stabilization of MPs.
However, there are some differences between MSP Nanodisc and Synthetic Nanodisc (as shown in table).
MSP Nanodisc Protein

Synthetic Nanodisc Protein

StabilizerMSP

PolymerSize

7-17nmAround 10nm

Wuhan institute of Biotechnology B7, Biolake No.666 Gaoxin Road, Wuhan, Hubei, China 430074+86-400-006-0995
info@dimabio.com
www.dimabio.com
6

DIMA Biotechnology LTD

Lipid composition

Artificial phospholipid environ
ments

Native cell membrane lipids

Proteins obtained fromProtein sources

Cell-free reaction or

Directly from the cells

detergent-solubilized method
Involvement of deterge

May use detergent duringnt

protein preparation

No detergent

MSP Nanodisc protein is uniform in size, making it a perfect tool for applications such as Cryo-EM,
whereas additional screening step may be required for the polymer Nanodisc proteins because the diameterof the polymers used for this type of Nanodisc protein is variable.
One major challenge associated with MSP Nanodisc protein is the interference caused by the presence ofMSP protein. The amount of target protein can’t be easily determined by measuring the absorbance of thesolution at 280nm for MSP Nanodisc protein. Moreover, MSP can lead to background noise when used
together with target protein in different assays.
For MSP Nanodisc proteins, due to detergents involved in the process of dissolving the cell membrane, it isnecessary to select a detergent that does not interfere with the folding of target protein. However, in
Synthetic Nanodisc proteins, the polymers act as both solubilizers and stabilizers, so no detergent isrequired.
Because of the benefits for Synthetic Nanodisc proteins, DIMA biotech developed an innovative
Synthetic Nanodisc platform for the full-length multi-pass transmembrane proteins.

5. Why do we use mammalian expression system to produce Nanodisc
proteins?
All DIMA’s proteins are prepared from mammalian cell expression system. Why?
Firstly, mammalian cells are known to accurately replicate the folding and post-translational modificationsof proteins, ensuring that the resulting nanodisc proteins closely resemble their natural counterparts. This isimportant for applications such as structural biology and drug discovery, where the native structure andWuhan institute of Biotechnology B7, Biolake No.666 Gaoxin Road, Wuhan, Hubei, China 430074+86-400-006-0995
info@dimabio.com
www.dimabio.com
7DIMA Biotechnology LTD
function of the proteins are crucial.
Secondly, mammalian cells provide a favorable environment for complex and multi-pass transmembraneproteins. These types of proteins often require specific chaperones and co-factors for correct folding andassembly, which can be more easily achieved in mammalian cells compared to other expression systems.
This ensures that the resulting nanodisc proteins exhibit the desired functionality and stability.
Additionally, the use of mammalian cells allows for the incorporation of post-translational modifications,such as glycosylation, which are important for the biological activity of many proteins. These modifications
can affect the structure and function of Nanodisc proteins, making the mammalian expression system thepreferred choice for producing biologically relevant proteins.
Overall, the use of the mammalian expression system in DIMA’s production of Nanodisc proteins ensuresthat the proteins closely mimic their natural counterparts in terms of structure, function, andpost-translational modifications, making them ideal tools for a wide range of applications in biochemistry
and biomedicine.
Comparison of different expression hosts
Expression HostPros

Cons
Inclusion bodies

EconomicE.coli

Not for large protein
FastNo PTMs
FastYeast
Limited PTMs

Recommend Usage
Bacterial protein
Small proteins such as cytokine
and enzyme

No γ-carboxylation
Small proteins such as cytokine
N-glycosylation focusesand enzyme

on Mannose.
secretory protein

FastBaculovirus
Limited PTMs

No γ-carboxylation
N-glycosylation is very
simple without sialic acid.

cytoplasmic protein
toxic protein.

Wuhan institute of Biotechnology B7, Biolake No.666 Gaoxin Road, Wuhan, Hubei, China 430074+86-400-006-0995
info@dimabio.com
www.dimabio.com
8DIMA Biotechnology LTD
Complex MPs
diverse PTMs
Mammalian

Low yield

Recombinant Antibody

Good solubility
et al.

6. DIMA’s synthetic nanodisc
DiMProTM Synthetic Nanodisc is an innovative solution for multipass transmembrane proteins withfollowing advantages:
Full-length membrane proteins with high purity

High solubility in aqueous solutions

High stability, lyophilized powder stable at room temperatureProteins are in a native membrane environment and remain biologically active

No detergent, useful for cell-based assays



No MSP backbone proteins

The multipass transmembrane proteins produced by this technology can be used for various applications,
including ELISA, SPR affinity analysis, phage display screening, Immunization, cell based assays andprotein crystal structure analysis et al..

7. Featured Data from DIMA’s Synthetic Nanodisc Proteins
Human MDR-1 full length protein-synthetic nanodisc (FLP100029) 12 transmembrane protein

Figure 4. MDR-1-synthetic nanodisc validated data. (Left) Human MDR-1-Nanodisc, Flag Tag on
Wuhan institute of Biotechnology B7, Biolake No.666 Gaoxin Road, Wuhan, Hubei, China 430074+86-400-006-0995
info@dimabio.com
www.dimabio.com
9DIMA Biotechnology LTD
SDS-PAGE; (Right) MDR-1-Nanodisc (FLP100029) can bind anti-Flag monoclonal antibody and the EC50is 6.883ng/ml.


Human GPRC5D full length protein-synthetic nanodisc ( FLP100011) 7 transmembrane protein

Figure 5. GPRC5D-synthetic nanodisc validated data. (Left) Human GPRC5D-Nanodisc, Flag Tag on
SDS-PAGE; (Right) GPRC5D-Nanodisc (FLP100011) can bind anti-GPRC5D mAb (DME100090) and theEC50 is 32.86ng/ml.


Human CLDN6 full length protein-synthetic nanodisc (Cat. FLP100008) 4 transmembrane protein

Figure 6. CLDN6-synthetic nanodisc validated data. (Left) Human CLDN6-Nanodisc, Flag Tag on
SDS-PAGE; (Right) CLDN6-Nanodisc (FLP100008) can bind anti-CLDN6 mAb (BME100082) and theEC50 is 66.99ng/ml.
Click to view all of Synthetic nanodisc proteins

References:Wuhan institute of Biotechnology B7, Biolake No.666 Gaoxin Road, Wuhan, Hubei, China 430074+86-400-006-0995
info@dimabio.com
www.dimabio.com
10DIMA Biotechnology LTD[1] Denisov, I., Sligar, S. Nanodiscs for structural and functional studies of membrane proteins. Nat Struct
Mol Biol 23, 481–486 (2016).
[2]Jonas A, Kezdy KE, Wald JH. Defined apolipoprotein A-I conformations in reconstituted high densitylipoprotein discs. J Biol Chem. 1989;264:4818–4824.
[3] Wald JH, Krul ES, Jonas A. Structure of apolipoprotein A-I in three homogeneous, reconstituted highdensity lipoprotein particles. J Biol Chem. 1990;265:20037–20043.
[4] Carlson JW, Jonas A, Sligar SG. Imaging and manipulation of high-density lipoproteins. Biophys J.
1997;73:1184–1189.
[5] Schuler MA, Denisov IG, Sligar SG. Nanodiscs as a new tool to examine lipid-protein interactions.
Methods Mol Biol. 2013;974:415-33.
[6] Jo M, Jung ST. Engineering therapeutic antibodies targeting G-protein-coupled receptors. Exp Mol
Med. 2016 Feb 5;48(2):e207.
[7] Reis RI, Moraes I. Probing Membrane Protein Assembly into Nanodiscs by In Situ Dynamic Light
Scattering: A2A Receptor as a Case Study. Biology (Basel). 2020 Nov 13;9(11):400.

Wuhan institute of Biotechnology B7, Biolake No.666 Gaoxin Road, Wuhan, Hubei, China 430074+86-400-006-0995
info@dimabio.com
www.dimabio.com
11