Purification of Apolipoprotein A-1 For the Synthesis of Nanodiscs in the Structural Study of Bcl-2 Family Membrane Proteins

Nanodiscs are a novel model membrane system, ideal for the structural biology of the membrane proteins.  Nanodiscs are discoidal phospholipid bilayers bounded by a membrane scaffold protein; such as Apo A-1 and its derivatives.  The purification of apolipoprotein A-1 is essential for the synthesis of Nanodiscs.  For protein expression, the apoA-1 cDNA was subcloned into the pET20b+ plasmid (Novagen, Madison, WI) to yield the plasmid construct, pNFX-pET20b (Provided by John Voss Ph.D. of UC Davis).  The plasmid was propagated in the XL1 blue cells (for storage), purified using a DNA purification system, and analyzed by DNA sequencing (Sequencing facility at University of Chicago).  After confirming the DNA sequence coding for the Apo A-1 protein, the plasmid was transformed into the expression host, BL21(DE3)pLys E. coli cells.  The protein was over-expressed by induction using IPTG (isopropyl thiogalactoside) and confirmed by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) analysis.  The protein will be further purified using a Hi-Trap affinity column chromatography.  Nanodiscs will be formed by reconstituting the purified protein with different lipids that mimic the native membrane environment of the Bcl family proteins. Subsequently, Nanodiscs will be used to perform structural studies of the Bcl-2 family membrane proteins.