They are much larger than typical epitope tags. “Multifunctional GFP” (mfGFP) variants that include multiple affinity tags within an internal loop of the EGFP were engineered to be used as both reporters and tags. GFP, green fluorescent protein, and its variants like EGFP, Clover are the most popular reporter proteins they have also been used as tags, for example, as a reporter for TP53 MITE-seq screen or when linked with LC3, as a reporter for autophagy. Purification: Cytiva Ni Sepharose Bio-Rad Nuvia IMAC resin Thermo Fisher HisPur GE Healthcare HisTrap HP Īntibody:SCBT sc-40, Abcam ab32, ab24609, Thermo Fisher, Cell Signaling, ICL Īntibody: Thermo Fisher ( R960-25), Cell Signaling Technology Purification: GE Healthcare GS4B Thermo Fisher Scientific 16101 CST (11847S) Īntibody: Novus Biologicals, Miltenyi Biotech These two technologies work together to clearly provide a solution to meet the challenge of expressing membrane proteins.Antibody: MilliporeSigma ATCC HB-9259 (clone 4E11) MBL CST 14793 48 hours or more post-transfection, the VLPs containing the membrane protein of interest can be harvested by centrifuging the culture medium to remove cells and cell debris, then precipitating the VLPs from the clarified culture supernatant and resuspending them in PBS.īy combining the capabilities of the high expression Expi293 system and MembranePro technology researchers can obtain greater than 20-fold increases in membrane protein yield compared to traditional adherent culture protein expression and up to 30-fold enrichment of the membrane protein on the surface of the VLPs compared to membrane fractions (Figure 3). Expi293F cells in Expi293™ expression medium are transfected with a plasmid expressing the membrane protein of interest and the MembranePro reagent according to the recommended protocol using ExpiFectamine 293 reagent. The VLPs are easily collected from the culture medium, providing a workflow that is much less labor intensive than preparation of traditional membrane fractions (Figure 2).įigure 2.
This enables these proteins to be displayed in their native context in a highly enriched nano-lipoparticle. MembranePro technology delivers overexpressed membrane proteins on virus-like particles (VLPs) that self-assemble and bud off the cell surface. All components work in concert to additively increase expression. The key features that bring about the superior protein yields of the Expi293 system are the enriched Expi293 Expression Medium, which supports high density culture of superior productivity Expi293F cells, and transient expression powered by the novel ExpiFectamine 293 transfection reagent and enhancers, which are designed to work specifically under high-density culture conditions. All yields are given as mg of protein per L of culture transfected (mg/L).Įxpi293 MembranePro Expression System provides superior yields of difficult to express membrane proteins Protein was harvested at day 5 to day 7 post-transfection. Expression was performed in 1ml, 30mL and 1 liter cultures. Expression of human erythropoietin (EPO), crypto and a human IgG in the FreeStyle 293 system (light columns) compared to expression in Expi293 (dark columns). We have observed expression levels of greater than 1 gram per liter of culture for some proteins (Figure 1).įigure 1. Typical Expi293 yields are 2- to 10-fold higher than other options. The Expi293 Expression system is a rapid, scalable system that delivers the highest protein yields of any current transient expression method. Rapid and ultra high-yield protein production in mammalian cells
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