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.
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).
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.
CD20 (CSB-MP015007HU) (4 times) is detected by Mouse anti-6*His monoclonal antibody.
The VLP-like structures of CD20 (CSB-MP015007HU) (4 times) has been confirmed by TEM.
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.
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.
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.
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.
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.
A: There are many, but it involves customers projects, it is inconvenient to inform.
A: Relatively stable, and the specifics are also related to each project (the specific data is temporarily confidential).
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).
A: We can provide empty VLPs as control for free, the negative control value is 0.1 or lower.
A: The envelope of VLPs is derived from the cell membrane and itself is a stable membrane.
A: The three bands coorespond to monomer, dimer and trimer respectively.
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.
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.
A: Yes, the structure are identical except for CCR4.
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.
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.
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.
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.
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.
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.