Frequently Asked Questions
The Lipoparticle
Shotgun Mutagenesis
Q: What are Lipoparticles?
A: Lipoparticles are non-infectious virus-like particles (VLPs) containing structurally-intact membrane proteins of interest on their surface.
Q: What is inside the Lipoparticle?
A: The Lipoparticle interior is composed of a well-defined polyprotein (Gag) that drives the production of Lipoparticles within cells. Gag is necessary and sufficient for Lipoparticle production. Cellular cytoplasmic proteins are generally not incorporated into Lipoparticles.
Q: How many receptors are incorporated onto Lipoparticles?
A: Lipoparticles typically contain 50-200 pmol/mg of specific receptor, which translates into approximately 50-200 receptors per Lipoparticle. Receptor incorporation is directly related to the expression level of the receptor at the plasma membrane.
Q: Are receptors on Lipoparticles conformationally intact?
A: Receptors are incorporated onto Lipoparticles directly from the cell surface and are never extracted or refolded, so are conformationally intact. Lipoparticles are produced from eukaryotic cell lines so receptors also contain native post-translational modifications. Conformational integrity is demonstrated using conformation-dependent antibodies, ligand binding, and/or functional assays, depending on the receptor and available assays and reagents.
Q: Do Lipoparticles contain non-specific membrane proteins?
A: Lipoparticles bud from the cell surface, so contain specific membrane proteins of interest as well as additional membrane proteins from the cells used for production. The effects of non-specific membrane proteins are minimized by over-expression of the membrane protein of interest, the use of alternative cell types where necessary, and the use of null particles that do not contain the membrane protein of interest as negative controls.
Q: What membrane proteins can and cannot be incorporated into Lipoparticles?
A: All plasma membrane proteins tested to date can be incorporated into Lipoparticles, although cellular expression levels influence Lipoparticle incorporation levels. Membrane proteins that are not expressed on the cell surface are not incorporated.
Incorporation of the target membrane protein is characterized during initial production optimization experiments.
Q: Can complexes of membrane proteins (e.g. 2 or more) be incorporated?
A: Yes, two or more membrane proteins can be incorporated simultaneously, as needed.
Q: Are Lipoparticle lipid membranes different than cellular lipid membranes?
A: Lipoparticle membranes are similar to lipid raft regions of cellular membranes, and are enriched in cholesterol and sphingolipids.
Q: How stable are Lipoparticles?
A: Lipoparticles should be treated similar to other purified proteins. Lipoparticles are stable for at least 6 months when stored refrigerated or frozen, and are stable in the presence of small amounts (<5%) of common additives (e.g. DMSO, ethanol). Like most lipid structures, Lipoparticles will be disrupted by detergents. Lipoparticle suspensions will appear as an opaque solution and should be gently mixed prior to use. Excessive vortexing of Lipoparticles should be avoided. Excessive dilution or filtration of Lipoparticles may cause sample loss.
Q: Are Lipoparticles safe?
A: Lipoparticles are not viruses. Although they are made using a viral protein, Lipoparticles lack numerous components that would be required for infection, replication, or integration, including a genome, an Envelope protein, LTR regions, and many enzymatic proteins. They should be treated similar to other purified proteins of biological origin. Lipoparticles include proteins derived from a retrovirus that is not known to infect or cause disease in humans. Nevertheless, Lipoparticles are derived from biological sources and should be handled with caution within a biological laboratory environment. Lipoparticles are not to be used in humans.
Q: Can I modify or label Lipoparticles?
A: Lipoparticles are non-living biochemical reagents, so can readily be modified with biochemical or fluorescent labels, either during production or after purification.
Q: How can I try Lipoparticles?
A: Contact us to purchase Lipoparticle evaluation samples. Once you have completed your evaluation, we can custom produce Lipoparticles containing your own receptor of interest.
Q: What is Shotgun Mutagenesis?
A: Shotgun Mutagenesis is Integral Molecular’s proprietary strategy for rapid structure-function mapping. Every residue in a desired protein is changed at least once, and each mutant protein is analyzed for function in eukaryotic cells.
Q: What kinds of proteins can be tested?
A: Any desired protein or DNA sequence that can be expressed within an animal cell line can be assayed using Shotgun Mutagenesis. Because of their difficulty and importance, Integral Molecular's first application of the technology is to GPCRs.
Q: What kinds of assays can be used in Shotgun Mutagenesis?
A: Any cell-based assay that is adapted to microplate format can be used for Shotgun Mutagenesis mapping. Examples include calcium-flux, viral infection, and immunofluorescence assays.
Q: What is needed to start?
A: Tell us the gene you would like to start with and we will clone, mutate, sequence, verify, and analyze it for you. If you would like the gene analyzed for its interaction with a specific ligand (e.g. antibody, drug, or natural ligand), you will need to provide it.
Q: What results from Shotgun Mutagenesis mapping analysis?
A: At the end of the analysis, you will receive the raw data from the experiments, a list of each amino acid in the protein scored for its importance in the interaction, and a model diagram of the protein with critical residues highlighted.
