DirectMX – One-Step Reconstitution of Membrane Proteins From Crude Cell Membranes Into Salipro Nanoparticles
Authors: Robin Löving
Liuhong Chen
Michael J Skynner
Stefan Klinter
Pilar Lloris-Garcerá
Jens Frauenfeld
Journal: Frontiers in Bioengineering and Biotechnology (2020)
Discipline: Multidisciplinary
Keywords: DirectMX; GPCR; SLC transporter; Salipro nanoparticles; direct membrane extraction
Affiliations: Salipro Biotech AB, Stockholm, Sweden., Bicycle Therapeutics, Cambridge, United Kingdom.

Abstract:  Integral membrane proteins (IMPs) are central to many physiological processes and represent ∼60% of current drug targets. An intricate interplay with the lipid molecules in the cell membrane is known to influence the stability, structure and function of IMPs. Detergents are commonly used to solubilize and extract IMPs from cell membranes. However, due to the loss of the lipid environment, IMPs usually tend to be unstable and lose function in the continuous presence of detergent. To overcome this problem, various technologies have been developed, including protein engineering by mutagenesis to improve IMP stability, as well as methods to reconstitute IMPs into detergent-free entities, such as nanodiscs based on apolipoprotein A or its membrane scaffold protein (MSP) derivatives, amphipols, and styrene-maleic acid copolymer-lipid particles (SMALPs). Although significant progress has been made in this field, working with inherently unstable human IMP targets (e.g., GPCRs, ion channels and transporters) remains a challenging task. Here, we present a novel methodology, termed DirectMX (for direct membrane extraction), taking advantage of the saposin-lipoprotein (Salipro) nanoparticle technology to reconstitute fragile IMPs directly from human crude cell membranes. We demonstrate the applicability of the DirectMX methodology by the reconstitution of a human solute carrier transporter and a wild-type GPCR belonging to the human chemokine receptor (CKR) family. We envision that DirectMX bears the potential to enable studies of IMPs that so far remained inaccessible to other solubilization, stabilization or reconstitution methods. 

Retracted: No

PubPeer comments:
 There is a significant need for reliable and reproducible methods for incorporation of intergral membrane proteins into supported, detergent free structures that retain the native plasma membrane lipid environment. While the method outlined in this publication is potentially interesting the manuscript is misleading and fails to include some important basic information as well as important controls. This method is, in fact, niether direct of one-step: crude membranes are first solubilised in digitonin before being incorporated into the (human) Saposin A (SapA) support. There is no data that supports the claim that native membrane are also incoporated, the particles produced could be entirely composed of the target protein surrounded by digitonin. The model GPCR used in the study is not named, which is not appropriate for a peer reviewed publication. More importantly there is NO data to demonstrate that the SapA encapsulated protein retains function, no ligand binding or G protein activation is presented so the utility of the proposed method can t be assessed. No indication of reproducibility is provided. ,