Creative Biostructure can provide custom Mempro™ phosphatidylinositol (PI) 3-/4-kinase production services using detergent-free expression system.
Phosphatidylinositols can form the component on the cytosolic side of eukaryotic cell membranes. PI 3-kinases are a family of enzymes that have important roles in cellular functions such as cell growth, proliferation, differentiation, motility, survival and intracellular trafficking. The products of PI 3-kinase include PI-3-P, PI-3,4-P(2) and PI-3,4,5-P(3), which function as secondary messengers in cell signalling. PI 4-kinases are a family of enzymes that act on PI in the first committed step in the production of the secondary messenger inositol-1'4'5'-trisphosphate. Conventional methodology for membrane protein production require extraction from the membrane into detergent micelles. However, the significant obstacle of detergent is that can induce conformational changes on the proteins. Our alternative detergent-free expression system can best preserve the native conformation of PI 3-/4-kinases for further functional analysis.
Figure 1. Structural model of PI 3-kinase catalytic subunit type 3. (OPM database)
Creative Biostructure has extensive expertise in high-yield PI 3-/4-kinase production in detergent-free protein expression system, we can employ various strategies for Mempro™ detergent-free PI 3-/4-kinase production, including:
Nanodiscs are the self-assembled technique to stabilize transmembrane proteins removed from the membrane by membrane scaffold proteins (MSPs) without detergent. Creative Biostructure can reconstitute PI 3-/4-kinases into nanodiscs in detergent-free procedure: Expressing the PI 3-/4-kinase in the cell-free expression system with the addition of pre-assembled nanodiscs.
The amphipathic amphipols have the ability to enwrap transmembrane proteins, allowing them to stay folded. Creative Biostructure can apply A8-35 to solubilize PI 3-/4-kinases during purification and stabilization processes.
The SMALPs can be self-assembled by the simple addition of the SMA co-polymers. At neutral or alkaline pH, The disc-like structure assembles itself, encapsulating PI 3-/4-kinases in a formation amenable to be purified.
These novel detergent-free techniques for membrane protein production can be obtained easily, and enabling more comprehensively structural and functional studies.
Creative Biostructure provides other various Mempro™ membrane protein production services. Please feel free to contact us for a detailed quote.
References:C. Tribet, et al. (1996). Amphipols: Polymers that keep membrane proteins soluble in aqueous solutions. Proc Natl Acad Sci U. S. A. 93: 15047-15050. J. Y. Jung, et al. (2002). Phosphatidylinositol 3- and 4-phosphate are required for normal stomatal movements. Plant Cell., 14(10): 2399-2412.M. Jamshad, et al. (2011). Surfactant-free purification of membrane proteins with intact native membrane environment. Biochem. Soc. Trans., 39: 813-818.M. Fujimoto, et al. (2015). Phosphatidylinositol 3-kinase and 4-kinase have distinct roles in intracellular trafficking of cellulose synthase complexes in Arabidopsis thaliana. Plant Cell Physiol., 56(2): 287-298.