Transmembrane protein (TP), also known as intact protein, is a type of membrane protein exists in the whole biofilm. Transmembrane protein plays an important role in basic physiological processes, including molecule transport, signal transduction, energy utilization, etc. On one hand, they are useful for signaling to the cell what the external environment contains. On the other hand, they serve as a way for the cell to gather information about the external environment.
Almost 30% of genes in genomic sequence encode transmembrane proteins, of which 50% are targets for currently known drugs. However, expression and purification of transmembrane protein are very difficult. And CUSABIO will help you to overcome the difficulties.
● Multiple membrane protein expression technology platforms.
Virus Like Particles (VLPs) Platform
During the expression process of enveloped virus capsid protein, it can self-assemble into nanoparticles. After the expression of the target protein (transmembrane protein), it is located on the cell membrane, and VLP is released by budding. The transmembrane proteins are displayed on the envelope of the released nanoparticles (ie, VLPs particles).
In this platform, CUSABIO mainly use the mammalian cell expression system for target protein expression. The post-translational modification of the mammalian cell system is the closest to the natural protein, and most pharmaceutical companies choose the drug target proteins from human species, so the Mammalian cell system is the best choice.
Advantages:
Full-length TPs with complete natural conformation.
Higher immunogenicity.
Higher abundance than that of overexpressing cells.
Can be the best targets for phage display because of their 100-200nm in size.
Can be used in immunization/ELISA/SPR/BLI.
Detergent Micelle Platform
Because of their strong hydrophobicity, transmembrane proteins are either difficult to dissolve in conventional solvents or cannot display the correct conformation. Appropriate detergents need to be added to aid solubility. CUSABIO has specially built a detergent technology platform based on cell free expression. Synthesize proteins by adding DNA templates, ATP, amino acids, various substrates, and enzymes derived from cell extracts in vitro, and then extract the target protein by screening different detergents.
In this platform, CUSABIO uses in vitro E.coli expression system for the target protein expression, then by screening different detergents to extract the target protein.
Advantages:
Can express the full-length protein with multiple transmembrane domains, not limited to ECD epitopes.
Precise quantification, distinct from VLPs and Nanodisc platforms.
Can be applied to immunization/ELISA/SPR/BLI.
Nanodisc Platform
In the buffer containing detergents, most membrane proteins can maintain stability and activity. However, a few membrane proteins that are sensitive to detergents are not suitable for buffering conditions containing detergents, which are specifically incapable of purification and poor stability (easy to be degraded or obvious precipitation) and no activity. For this type of membrane protein, we suggest to try the nanodiscs technology.
In this platform, CUSABIO uses in vitro E.coli expression system for the target protein expression, the other expression systems such as mammalian cell system is also suitable, but we haven't developed yet. In the process of protein expression in the in vitro E.coli system, membrane skeleton protein (MSP) and phospholipid molecule (DMPC) were added to assemble nanodiscs during expression. Components: target protein (transmembrane protein), membrane skeleton protein (MSP), phospholipid molecule (DMPC).
Advantages:
Display natural conformation with complete biological activity.
Detergent-free, suitable for experiments that interfere with detergents.
Can be used directly for phage screening (provided empty nanodisc reverse screening).
Can be used for immunization/ELISA/SPR/BLI/cell assays.
CUSABIO, as an experienced protein manufacturer, provides the above three technology platforms to fuel the development of transmembrane proteins. Since establishment of these platforms, 200 proteins have been successfully produced with a yield of mg/ml, which contains 99 transmembrane proteins (TPs) with 1-15 transmembrane domains and toxic proteins that are difficult to express in traditional E.coli expression systems. We have also produced high molecular weight proteins (130 kDa -140 kDa) that contain multiple transmembrane domains.
● Nearly 400 full-length membrane proteins have been successfully developed.
CLDN18.2 (4 times)
CD20 (4 times)
ACKR1 (7 times)
ADIPOR1 (7 times)
RHD (11 times)
SLC7A11 (12 times)
● High purity determined by SDS-PAGE.
The purity of HTR1B (CSB-CF010882HUb2) (7 times) is greater than 90% as determined by SDS-PAGE with 5% enrichment gel and 15% separation gel.
● High Specificity Validated by Western blot (WB).
CLDN18.2 (CSB-MP005498HU(A5)) (4 times) is detected by Mouse anti-6*His monoclonal antibody. The three bands respectively correspond to monomer, Homodimer, Homotrimer.
Measured by its binding ability in a functional ELISA. Immobilized human CLDN18.2 (CSB-MP005498HU(A5)) at 5 μg/ml can bind anti-CLDN18.2 recombinant Monoclonal Antibody (CSB-RA005498A1HU), the the EC50 is 5.225-9.256 ng/ml.
Measured by its binding ability in a functional ELISA. Immobilized Human CLDN6 at 10 μg/ml can bind Anti-CLDN6/9 recombinant antibody (CSB-RA005508MA1HU), the EC50 is 1.501-2.035 ng/mL
Measured by its binding ability in a functional ELISA. Immobilized human GPC3 (G537R) at 5 μg/ml can bind Anti-GPC3 recombinant antibody, the EC50 is 4.739-7.092 ng/ml.
Measured by its binding ability in a functional ELISA. Immobilized Human CCR4 at 10 μg/ml can bind Anti-CCR4 recombinant antibody (CSB-RA004843MA01HU), the EC50 is 362.3-630.8 ng/mL.
Measured by its binding ability in a functional ELISA. Immobilized Human GPRC5D at 5 μg/mL can bind Anti-GPRC5D recombinant antibody (CSB-RA882153MA01HU), the EC50 is 8.847-10.38 ng/mL.
Measured by its binding ability in a functional ELISA. Immobilized Human CXCR4 at 10 μg/ml can bind Anti-CXCR4 recombinant antibody (CSB-RA006254MA01HU), the EC50 is 101.7-253.6 ng/mL.
Measured by its binding ability in a functional ELISA. Immobilized human CCR8 (CSB-MP004847HU) at 5 μg/ml can bind Anti-CCR8 recombinant Antibody, the the EC50 is 11.13-17.29 ng/ml.
Measured by its binding ability in a functional ELISA. Immobilized human C5AR1 at 10 μg/ml can bind Anti-C5AR1 recombinant antibody (CSB-RA003996A0HU), the EC50 is 1.239-3.760 ng/mL.
FAQs
Q: How to choose a membrane protein technology platform?
A: Different membrane protein technology platforms have their own advantages and disadvantages. The selection should be based on the specific protein expression needs and application scenarios. You can consult with technical personnel from CUSABIO for specific guidance. Below is a comparison of different platforms:
Technology Platforms
Virus Like Particles (VLP) Platform
Detergent Micelle Platform
Nanodisc Platform
Coating for ELISA
Direct coating
Direct coating
Direct coating
Activity
Great
Good
Good
Stability
Most stable
Relatively stable
Relatively stable
Purity
Mixture
Relatively pure (only target protein)
Mixture
SPR/BLI
Detectable
Detectable (solution adjustment required)
Detectable
Factors to consider
Total protein concentration; relatively short preparation cycle
Relatively long preparation cycle
Relatively long preparation cycle
Q1: Are any viruses or viral vectors used in the preparation of membrane proteins with the VLP platform?
A: Mammalian enveloped VLPs are enveloped virus-like particles, which are self-assembled by one or several structural proteins of the virus into a virus-free genome that cannot replicate and have no infectivity, but are similar in shape and structure to complete viruses and do not contain viral nucleic acid substances. It’s safe. Now many vaccines will also choose this technology, which shows that it is safe.
Mammalian enveloped VLPs are co-transfected with two expression plasmids (expressing transmembrane protein and capsid protein respectively) without using any virus and viral vector, and the transfection reagents are used for transient transfection.
Q2: How many liposomes and membrane skeleton proteins combinations are selected for Nanodisc platform, and do you optimize the combination for each project?
A: In the early stage of platform development, a variety of membrane skeleton proteins and phospholipid molecules were tried, and orthogonal combinations were attempted, and finally a pair of optimal membrane skeleton proteins and phospholipid molecules was screened out. At present, the platform is mature and we use this same combination for different projects.
Q3: What is the coating concentration of VLPs and how much is used?
A: Considering that the total concentration of VLPs is measured, the recommended coating concentration is 2-10ug/ml, and the amount used for one well is 0.2-1ug.
Q4: What is the concentration of your VLPs?
A: The Bradford assay method is used for the concentration determination. The specific concentration of each item and each batch is different. Please consult the concentration information of the corresponding batch during pre-sales inquiry.
Q5: When VLPs is used as an antigen to immunize antibodies, does VLPs have any effect on the quality of antibodies, and the effect of different adjuvants?
A: At present, we have tested, without adjuvant, adding rapid adjuvant (without emulsification) and conventional Freund's adjuvant to evaluate the impact on immunity. From the final effect, it is recommended to use rapid adjuvant.
Q6: Have you successfully made a candidate antibody drug molecule using the protein developed from this platform?
A: There are many, but it involves customers projects, it is inconvenient to inform.
Q7: Is VLPs output production stable and what is the approximate level?
A: Relatively stable, and the specifics are also related to each project (the specific data is temporarily confidential).
Q8: If the customer's antibody does not recognize the protein, whether it belongs to the category of risk-free protection?
A: Considering the difference of different positive antibodies, we will generally produce a positive antibody by ourselves. If both of the customer's antibody and our produced positive antibody don’t bind, it belongs to the risk-free category (no charge for inactivity).
Q9: Do you provide empty VLPS as a control? In your quality control data, what is the measured value of the empty VLPS negative control?
A: We can provide empty VLPs as control for free, the negative control value is 0.1 or lower.
Q10: Why can VLPs form stable membranes?
A: The envelope of VLPs is derived from the cell membrane and itself is a stable membrane.
Q11: Why does the WB image of Claudin18.2 have three bands?
A: The three bands coorespond to monomer, dimer and trimer respectively.
Q12: Can you please share any tips regrading safe detergents and its concentrations that I can use to extract the recombinant proteins from the VLP particles?
A: We are sorry to tell you that VLPs and target protein cannot be separated, because VLPs protein is an enveloped virus-like particles, and the target protein is displayed on the surface. The target protein can be close to the natural conformation in VLPs. So the VLPs protein also cannot be accurately quantified. But we provide the VLPs control protein under the same preparation conditions for free to the customer to deduct the influence of the host membrane protein on the envelope.
Q13: Regarding the Virus-Like Particles (VLPs) Mammalian cell expression system , could you please let me know in which buffer the protein is provided ? Also as it is membrane protein, is the protein supplied in liposome, or will it be solubilized?
A: The standard buffer that we normally use is PBS, pH 7.4, 6% trehalose. We can etither provide it in liquid form or lyophilized form, please communicate with us in advance if you have any special requirement for the buffer or the shipping format.
Liquid form: It needs to be shipped with dry ice and we need to charge extra fees for dry ice and dry ice box.
Lyophilized form: It could be shipped with normal bule ice packs and no extra charges. However, if you request to ship with dry ice, please communicate with us in advance and extra fees will be charged.
Transmembrane proteins are displayed on the nanoparticle envelope in their native state.
Q14: Can we think the the structure of #CSB-MP3838 (Virus-Like Particles (VLPs) isotype control) and #CSB-MP004843HU are identical except for CCR4?
A: Yes, the structure are identical except for CCR4.
Q15: For immunization and antibody screening, which platform should I choose for Claudin18.2?
Our Claudin18.2 has been developed with VLP platform. Both of VLP version and the detergent version are suitable for immunization and screening. However, the VLP version may be preferred for improved immunogenicity. For accurate affinity measurements, maybe the detergent version is more suitable.
Q16: Why do you recommend to express the full-length transmembrane protein? After all, antibodies and antigens only bind on the extracellular domain.
Multi-pass transmembrane proteins span the cell membrane multiple times forming multiple extracellular domains. For example, Claudin18.2 and CD20 have two extracellular loops (ECLs) with each ECL having specific functions and interactions with each other. Full length can ensure that the protein conformation is biologically relevant while enabling the ECL to be completely exposed for improved screening of ideal antibodies. According to our experience, the full length is active and relevant compared to isolated extracellular domain. It is not the case that CUSABIO always emphasizes full length proteins, but drug discovery R&D work needs the full-length multi-pass transmembrane proteins. Indeed, when compared, the activity of only the ECL region is worse than that of the full-length protein. CUSABIO is committed to providing the high quality and relevant products that meet customers' needs.
17: How do you confirm that Claudin18.2-VLP contains the target protein and how do you evaluate its purity? In the EM image on your product page, how do you tell whether the protein is embedded on the VLP particle?
Claudin18.2-VLP is tested and verified by anti-Claudin18.2 specific antibodies. The existence is evaluated by WB/SEM. The conventional negative dye EM cannot see whether Claudin18.2 is on the VLP due to its low resolution. Binding activity with anti-Claudin18.2 specific antibodies can confirm the presence of Claudin18.2 on the particles.
Q18: Are you able to provide VLP-membrane proteins in lyophilized form? Could it be emulsified, and are there any requirements for the type of adjuvant? What are the precautions for using VLP formulated proteins for immunization?
After our continuous technical optimization, we can now provide VLP-membrane proteins in lyophilized form, which can be transported by normal blue ice packs. VLP immunization mainly requires attention to the choice of adjuvant dose, because VLP itself can enhance immunogenicity, which is not the case for conventional protein products.
Q19：How long does it take for the VLP platform to develop successfully?
A: Generally, it will take 1.5 to 2.5 months, including the activity test. If customers have positive antibodies, they can send us to test activity.
Useful Resources
Transmembrane proteins are located at the interface between cells and the outside world, mediating the signal transduction between cells and the outside world, and performing many important cellular biological functions. For example, it is a receptor for various signaling molecules, hormones and other substrates; it is involved in the exchange of substances, energy and signal between the inside and outside of the cell membrane; it constitutes a channel for various ion transmembranes, which inputs nutrients and some inorganic electrolytes into cells, and discharges toxic or useless metabolites into cells.
Transmembrane protein products expressed by cell-free expression systems can be used to study the functions of transmembrane proteins. The integration of transmembrane proteins into vesicles for structural and functional studies is a hot topic in membrane protein research.
According to its structure, transmembrane proteins can be classified into alpha helix and β - barrel membrane proteins. For details, please refer to the article: A Resume for Transmembrane Proteins.
-SDS.jpg)
-WB.jpg)
-TEM1.jpg)
-AC1.jpg)
-AC1.jpg)
-AC1.jpg)
-AC1.jpg)
