Obtaining Access To Membrane Proteins Using Cell-free Technologies And Nanolipoproteins

We have developed a novel process for the production and assembly of nanolipoprotein particles (NLPs) as a reagent for imaging, drug delivery, immunomodulation and stabilizing membrane protein complexes. Nanolipoproteins (NLPs), also referred to as Nanodisc, were first described as components of the human HDL lipoprotein complexes that could be isolated and reconstituted to form NLPs.  NLPs are discoidal nanoparticles of 10 – 20 nm that self-assemble around a phospholipid bilayer capable of supporting membrane proteins. This bilayer mimics closely the cell membrane and can support small molecules such as dyes and proteins. NLPs present distinct advantages in terms of particle size, monodispersity and consistency: the presence of the circular protein belt constrains the dimensions of the bilayer and ensures NLP particle size. NLPs have shown great promise as a biotechnology platform for solubilizing and characterizing membrane proteins. By the simple addition of a few constituents to cell-free extracts, this approach provides a rapid process for the production of functional soluble membrane protein complexes that eliminates the need for cell growth, lysis, and subsequent purification. We have demonstrated this process on multiple membrane proteins important for rhodopsin-related proton pumps, drug transporters, host-pathogen interactions, herceptin receptor family and G-coupled protein receptors. Proteins have ranged in size from 10 – 200 kDa, with 2 or more transmembrane domains and have been shown to be biochemically functional. Our approach represents a solution to the challenge of generating soluble functional membrane proteins, facilitating the structural and functional characterization of these critical, yet poorly understood molecules.