Poster #034, University of California, San Francisco
Expression and Purification of MSPs and the Making of Nanodiscs
Mentors: Sourobh Maji, PhD and Robert Stroud, PhD, MA
MSP1E1D1 and MSP1E3D1 are Membrane Scaffold Proteins (MSPs). These are genetically engineered from the Apolipoprotein A-I. They are used for the construction of lipid nanodiscs for the study of membrane proteins since they provide a native-like environment that mimics the cellular bilayer and eliminates the need for destabilizing chemical detergents. With this research we aim to express and purify MSP1E1D1 and MSP1E3D1 for nanodisc production. We transformed Escherichia coli cells with plasmid carrying kanamycin resistance and target MSPs. Following transformation, we increased a primary starter culture into a larger secondary culture. Protein expression of the MSPs was induced using isopropyl β-D-1-thiogalactopyranoside. The cells were centrifuged and harvested, then lysed using an Emulsiflex, and cleared of cellular debris. The target proteins, MSP1E3D1 and MSP1E1D1, were isolated from the crude lysate using Nickel-Nitrilotriacetic Acid (Ni-NTA) affinity chromatography. We did a SDS-PAGE gel electrophoresis to confirm purity and mass of proteins. Finally, the purified proteins were concentrated and isolated via Superdex 75 size exclusion chromatography to isolate MSP particles. After finalizing our overall protein purification our results show that we successfully isolated high-quality MSP1E1D1 and MSP1E3D1 proteins at a concentration of 6mg/ml. Therefore, we can proceed to use the extracted proteins to assemble our desired nanodiscs for membrane protein study.