Membrane Protein Lentivirus
Creative Biolabs has developed a comprehensive list of membrane protein tools to support the development of antibody and other therapeutic. We are dedicated to bringing more value to our clients by providing stock products, more technical resources and custom protein production services.
Background of Membrane Protein
About one-third of all proteins in living things are membrane proteins. Membrane proteins can be divided into three groups based on their structural characteristics: integral membrane proteins, which are permanently anchored to or a part of the membrane; peripheral membrane proteins, which are momentarily attached to the lipid bilayer or to other integral proteins; and lipid-anchored proteins. Membrane proteins, such as membrane receptors, ion channels, GPCRs (G protein-coupled receptors), and different transport proteins, play important functions in all organisms. membrane receptors, which are located in cell membranes and can communicate with both the cell's internal environment and its exterior environment. Some enzymes, such oxidoreductase, transferase, and hydrolase, are also membrane proteins. Cell adhesion molecules, which are found on the cell surface and involved in interacting with extracellular matrix (ECM) or other cells, enable cells to recognize and communicate with one another.
Application of Membrane Protein Lentivirus
The lentivirus produced by Creative Biolabs are enveloped viruses that enter cells by fusing with membranes. Our lentivirus is made up of vector particles containing glycoproteins from other enveloped viruses, specifically the vesicular stomatitis virus (VSV-G). Given the stability and extensive host range that VSV-G pseudotyping offers, these virus particles are very helpful for attacking many cell types.
Feature of Membrane Protein Lentivirus
- Infect both actively-dividing and non-dividing cells.
- Infect a variety of cell stages.
- Ideal for transducing stem cells or primary cells that may be resistant to other transduction techniques.
- Up to 10 kb of DNA can be inserted.
- Long-lasting, reliable transgene expression.
Published Data
| Paper Title | Programmable extracellular vesicles for macromolecule delivery and genome modifications |
| Journal | Developmental Cell |
| Published | 2021 |
| Abstract | The passage of large macromolecules through the plasma membrane and endosomal barriers continues to represent a formidable obstacle. Using engineered ectosomes, researchers report a generalizable method for delivering proteins and ribonucleoproteins (RNPs) to cells in vitro and mouse liver tissue in vivo. Gectosomes are designed to co-encapsulate vesicular stomatitis virus G protein (VSV-G) with bioactive macromolecules via split GFP complementation. This method permits active cargo loading, improves the specific activity of cargo delivery, and facilitates Gectosome purification, as determined by our findings. Active cargo loading reduces the non-specific encapsulation of cellular proteins, especially nucleic acid-binding proteins, according to analyses based on experiments and mathematical models. Using Gectosomes encapsulating Cre, Ago2, and SaCas9, they demonstrate their ability to execute designed modifications of endogenous genes in cell lines in vitro and mouse liver tissue in vivo, paving the way for the use of this technology in the treatment of a variety of human diseases. |
| Result |
They attempted to separate Gectosomes from other EVs using differential ultracentrifugation (UC) and density gradient flotation. Even though this procedure effectively enriches exosomes as measured by CD9 and GM130, Gectosomes components are also enriched in 100-K UC sediments. To address this issue, they developed a four-step Gectosome purification procedure that is scalable. After two differential centrifugation steps, the resuspended 10K pellet was applied to a qEV 70 nm (IZON) size SEC column, and fractions were collected using FITC fluorescence. Gectosomes were captured using magnetic beads immobilized with anti-VSV-G 8G5F11 antibody prior to final elution with low pH glycine. The majority of Gectosomes are present in the second and third qEV fractionations based on fluorescence intensity and western blot analysis of VSV-G-GFP11 and Cre-GFP1-10. VSV-G to Cre ratios in fraction 2/3 are significantly higher than those in the UC sample. While 100,000 x g ultracentrifugation fractionation produces highly enriched CD9 and GM130, IZON fractionation followed by immunocapture decreases CD9 and GM130 while increasing VSV-G in fractions 2 and 3, relative to the respective proteins in UC samples. The remaining CD9 in the Gectosome fractions may originate from the cell surface, as indicated by this result. This result provides additional support for the biochemical distinction between Gectosomes.
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Fig.1 Purification, quantification, and mathematical modeling of Gectosomes. (Zhang, 2021)
Reference
- Zhang, X.J.; et al. Programmable extracellular vesicles for macromolecule delivery and genome modifications. Developmental Cell. 2021, 55: 1-18.
