Developing a Purification System to Assess SR‐BI Structure and Function

High density lipoprotein (HDL) is considered anti-atherogenic due to its ability to remove cholesterol from the periphery and deliver it to the liver via its receptor, scavenger receptor BI (SR-BI). The interaction between HDL and SR-BI is the most important mechanism to remove bodily cholesterol. SR-BI is crucial for maintaining lipid homeostasis, as humans with SR-BI mutations display impaired cholesterol clearance and an elevated risk of cardiovascular disease (CVD). As there currently exists no full-length structure for any of the Class B scavenger receptors, the goal of this study is to resolve a high-resolution structure of functional full-length SR-BI. This structure would be a pivotal addition to the scientific toolbox employed to uncover questions of cholesterol transport in the context of cardiovascular disease. As first steps in the purification process, suspended sf9 cells were infected with baculovirus containing human SR-BI-encoding bacmid. After 96 hours post-infection, cells expressing SR-BI were centrifuged, lysed, and membrane proteins were solubilized into mixed micelles. SR-BI within micelles was then purified through a series of steps that included affinity column purification, desalting, and concentration. Upon successful purification, verifying the expression of functional SR-BI at the cellular surface was a critical step. To assess SR-BI function, sf9 cells were infected with baculovirus containing SR-BI encoding bacmid or empty vector and plated. Expression of human full-length SR-BI significantly increased HDL cellular association and uptake of HDL-cholesteryl esters compared to uninfected and empty vector infected cells. Additionally, free cholesterol efflux and membrane cholesterol distribution were increased with SR-BI expression. Lastly, SR-BI expressed in sf9 cells maintained its ability to form higher order oligomers, suggesting synthesized receptor displays its expected functions. This purification protocol and functional assays represent the first steps in resolving the first high-resolution structure of human full-length SR-BI and provide promise for delineating the ways in which the HDL/SR-BI relationship allows for efficient cholesterol clearance.