RESUMO
The laboratory protocol presented here takes about 3 hours to perform and investigates protein and lipid interactions. Students first purify His6 -tagged human apolipoprotein A-I (apoA-I) with Ni-NTA affinity resin in a simple batch protocol and prepare multilamellar vesicles (MLV) from pre-dried phospholipid films. When apoA-I is added to the MLV, much smaller protein/lipid nanodisc complexes are formed in some instances. Nanodisc formation can be monitored by a decrease in light-scattering intensity at 340 nm using a simple spectrophotometer. Students will observe nanodisc formation with MLV formed from the anionic phospholipid dimyristoylphosphatidyl glycerol, which pack poorly into lipid bilayers, but not with MLV formed from the zwitterionic phospholipid dimyristoyl phosphatidylcholine, which form stable bilayers. This laboratory exercise is accompanied by questions and exercises that enable students a deeper of the dimensions of apoA-I and nanodiscs as well as the biological relevance of nanodisc formation in the process of reverse cholesterol transport.
