P-Glycoprotein: Purification, Incorporation and Activity in Nanodiscs

P-glycoprotein (P-gp), a member of the ATP Binding Cassette (ABC) superfamily, is a drug transporter that effluxes a broad spectrum of therapeutic agents. P-gp is expressed in many tissues important in drug disposition including the intestine, liver, kidneys, and blood-brain-barrier. P-gp acts to decrease drug absorption following oral administration, facilitate elimination from the body, and decrease drug exposure in tissues such as the brain. This 170 kDa protein consists of two similar halves, each composed of 6 helical transmembrane regions and an ATP-binding domain. Genetic variations in the ABCB1 gene that encodes P-gp lead to alterations in P-gp expression and activity, which can affect multidrug resistance and drug disposition. The goals of this study are to biochemically and biophysically characterize P-gp to understand how ABCB1 genetic polymorphisms alter activity. The first step in our study is to obtain functional protein incorporated into our model membrane system, the nanodisc. A nanodiscs is a discoidal system whose composition (lipid, cholesterol) can be custom tailored and is held in shape by two belt proteins, which also govern the size of the disc. This poster describes our path to producing active P-gp in nanodiscs using E. coli total lipids and two sizes of belt protein. The next step is to conduct studies to determine how ABCB1 genetic polymorphisms can lead to differences in transport activity and structural dynamics. Understanding these differences, as related to polymorphisms, can potentially lead to a method to reverse multidrug resistance.