Membrane Protein Expression in VLPs
Virus-like particles (VLPs) are hollow protein particles that are similar in shape to natural virus particles and are self-assembled by one or more structural proteins of viruses. The diameter of VLPs is about 20~200nm, which has a regular spatial structure and superior biocompatibility. The conformation of VLPs is very similar to that of real viruses, but it does not contain the nucleic acid required for virus replication and has no infective ability. Therefore, it does not have potential risks such as incomplete inactivation of virus, virus genome exchange and recombination, and virus atavism. Currently, VLPs have been widely applied for a variety of applications such as vaccines, antibody development, delivery systems, bioimaging, and cell targeting. Of note, VLPs have proven excellent utility in membrane protein display.
Beta Lifescience has developed Virus-Like Particles (VLPs) Technology based on our magic Membrane Protein Production platform to efficiently express and purify membrane proteins. Membrane Protein VLPs (MP-VLPs) are non-infectious virus-like particles (VLPs, about 150 nm in diameter) that display highly-expressed copies of specific membrane proteins in their native conformation. MP-VLPs are produced from host systems by co-expressing the retroviral structural core polyprotein (gag) and the target membrane protein. Retroviral gag core proteins self-assemble at the plasma membrane, where they bud from the host cells over-expressing a membrane protein of interest, enabling the formation of MP-VLPs.
Production Methods for the VLP Technology Platform
The appropriate choice of expression systems is one of the determinant factors in Membrane Protein Expression in VLPs. Beta Lifescience can perform VLPs Production by five following expression systems:
- Virus-like Particles (VLPs) Production in Bacterial Cells System: The bacterial cell system is the most widely used expression system for VLP production. This system is based on our well-characterized commercial Escherichia coli (E. coli) strains. The bacterial cell system not only enables VLPs to be produced in high yield and at low cost, but can also be easily scaled up. The main drawback of the bacterial cell system is the inability to perform post-translational modifications on recombinant proteins.
- Virus-like Particles (VLPs) Production in Mammalian Cells System: VLPs have shown good potential for the development of human vaccines and gene therapies. The mammalian cell system offers several advantages for VLP production. Compared with other expression systems, the mammalian cell system can not only improve production flexibility and stability, but also restore specific native glycosylation. Beta Lifescience can enable customized VLP production (particularly for influenza virus) using a wide range of mammalian cells, including HEK293 and CHO cells.
- Virus-like Particles (VLPs) Production in Yeast Cells System: The production of VLPs in yeast cells enables high-yield VLPs to be obtained in high-speed manufacturing. Compared with E. coli expression systems, yeast cells such as the Hansenula and Pichia strains are more complex, as they can select stable recombinants that contain the transgene integrated into the genome.
- Virus-like Particles (VLPs) Production in Insect Cells System: The insect cell system is another widely used system for VLP production, and offers the following advantages: rapid growth rate, easy scale-up capability and capacity for post-translational modification. The insect cell system can produce very complex VLPs, but the production yield is not very high.
- Virus-like Particles (VLPs) Production in Plant Cells System: The plant cell system is another attractive system, particularly for the production of VLP vaccines. VLPs in plant cells keep the manufacturing process low-cost, ensure appropriate post-translational modification and assembly, and reduce the risk of introducing adventitious human pathogens.
Advantages of Membrane Protein Expression in VLPs
- Ensure the natural conformation of transmembrane protein for many times, and improve the success rate of isolating antibodies that can recognize the structure of natural membrane protein.
- The abundance of target antigen in encapsulated VLPs is higher than that in over expressed cells.
- Higher immunogenicity
- Can be used as the best targets for dendritic cells and phage display in vivo because of their 100-300nm in size.
- Suitable for immunization/ELISA/SPR/BLI/cell experiment/CAR detection.
Service Details
VLP-based Membrane Protein Expression
Applications of Membrane Protein Expression in VLPs
VLPs present highly expressed copies of membrane proteins in their native conformation, providing an alternative to stable cell lines, crude membranes, detergent-solubilized proteins and other membrane protein preparation strategies (liposomes, nanodiscs). VLPs can be used for a wide range of applications.
- Immunization for antibody production
- Hybridoma screening for antibody discovery
- Phage and yeast display for antibody discovery
- Antibody characterization
- Membrane protein binding assays
- Membrane protein functional assays
Case Studies of Membrane Protein Expression
Utilizing the VLP membrane protein technology platform, Beta Lifescience has developed various full-length transmembrane proteins such as GPRC5D, Claudin 6, Claudin 18.2, and SSTR2. The activity of these membrane proteins has been verified.
Recombinant Human Claudin-18.2(CLDN18.2) - VLPs (Active)
Full-length Claudin18.2-VLP has been observed under an electron microscope to ensure that it is assembled correctly.
Western Blot: CLDN18.2-VLP is detected by Anti-Claudin 18 Antibody.
Measured by its binding ability in a functional ELISA. Immobilized human CLDN18.2 at 5 μg/ml can bind anti-CLDN18.2 recombinant Monoclonal Antibody, the the EC50 is 5.225-9.256 ng/ml.
Recombinant Human CD20 - VLPs (Active)
The purity of Human CD20 VLP is greater than 95% as determined by SEC-HPLC.
Immobilized Human CD20 VLP at 5μg/ml (100μl/Well) on plate on
the plate. Dose response curve for Rituximab, hFc Tag with the EC50 of 11.4ng/ml determined by ELISA.
Immobilized Human CD20 VLP at 5μg/ml (100μl/well) on the plate. Dose response curve for Obinutuzumab, hFc Tag with the EC50 of 1.7ng/ml determined by ELISA.
FAQs
The VLP platform uses HEK293 cells for expression, so the resulting protein is similar to native forms and more biologically relevant for further research and applications.
One T-175 flask yields approximately 500 µL of VLPs following resuspension. VLP protein concentration depends on protein expression and purification efficiency, and may be between 0.1 µg/µL and 1 µg/µL. Depending on the efficiency of expression of your GPCR and the activity of the GPCR for your ligand of interest, a single reaction may generate sufficient VLP sample for up to 1000 ligand binding assay data points.
Virus-like particles (VLPs) are a class of virus-like nanoscale particles with structures similar to real viruses but lacking viral genes.These particles are highly repetitive and organized, do not possess viral genetic material, do not replicate themselves, and are easy to infiltrate into cells. Thus possessing high biosafety，it is possible to display conformationally intact membrane proteins on the surface of the cell membrane on the VLP surface.
We use the BCA method to determine the concentration of VLP-based membrane protein products. If necessary, particle homogeneity of the VLP sample can be confirmed by SEC-HPLC or DLS to better identify the sample content.
Research Progress on VLP Systems for Membrane Protein Production
Virus-like particles (VLPs) show great promise for membrane protein production.VLPs provide stable environments that allow membrane proteins to maintain their functionality even after they are removed from their natural lipid bilayer. VLP technology has been applied to various membrane proteins, including those from SARS-CoV-2, influenza, and Zaire Ebola virus.
1. Efficient Expression and Proper Folding
VLP systems can efficiently express and properly fold membrane proteins in eukaryotic cells, providing a similar environment to native cells for complex post-translational modifications and folding mechanisms. Hepatitis B Virus-like Particles. The Hepatitis B surface antigen (HBsAg) is expressed in yeast or mammalian cells and self-assembles into VLPs, used in Hepatitis B vaccines such as Recombivax HB and Engerix-B[8].
2. Vaccine Development
VLPs are widely used in vaccine development due to their ability to mimic the structure of viruses, inducing strong immune responses while being non-infectious and safe[9]. Human Papillomavirus (HPV) Vaccines. The L1 protein of HPV is expressed in insect or yeast cells and self-assembles into VLPs, leading to the development of HPV vaccines like Gardasil and Cervarix, which effectively prevent HPV infections and related cancers. HIV Vaccine Research. HIV envelope proteins gp120 and gp41 are expressed in mammalian cells and self-assemble into VLPs, used in HIV vaccine research to induce strong immune responses and advance HIV vaccine development[10].
3. Drug Screening
Membrane proteins expressed in VLP systems can be used to create high-throughput screening platforms for potential drug molecules, particularly for G protein-coupled receptors (GPCRs) and ion channels. GPCRs are expressed and incorporated into VLPs in insect or mammalian cells, facilitating high-resolution structural biology studies and drug screening. For example, the β2-adrenergic receptor (β2AR) expressed in VLPs has aided in its structural analysis and ligand screening. Ion Channel Studies[11]. Ion channels such as voltage-gated sodium channels (Nav) are expressed in VLP systems, enabling drug screening and functional studies to understand their roles in nerve and muscle cells[12].
Future Prospects of VLP Systems for Membrane Protein Production
Virus-like particle (VLP) systems hold great promise for membrane protein production, covering potential breakthroughs in several fields. By providing a stable environment, VLP can maintain the functionality of membrane proteins, which is particularly important in the fields of drug discovery, structural biology, vaccine development, gene therapy and synthetic biology. In the future, optimization of the VLP production process will enhance the yield and quality of membrane proteins and enable large-scale production through advances in genetic engineering and expression systems. With the continuous research and development of VLP technology, more new functions and applications will be unlocked, making VLP a key platform for the study and application of membrane proteins and bringing new opportunities and challenges for biotechnology and pharmaceutical development.
Featured Virus-Like Particle (VLP) Proteins
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