Rapid automated detergent screening for the solubilization and purification of membrane proteins and complexes

Abstract

Membrane proteins constitute about one third of proteins encoded by all genomes, but only a small percentage have their structures deposited in the Protein Data Bank. One bottleneck in the pipeline from expression to structure determination is the identification of detergents that maintain the protein in a soluble, stable, and active state. Here, we describe a small‐scale automated procedure to easily and rapidly screen detergents for the solubilization and purification of membrane proteins, to perform detergent exchange, or to identify conditions preserving protein interactions in complexes. Hundreds of conditions can be tested in a few hours to select detergents that keep proteins folded and nonaggregated, from single membrane preparations of cells overexpressing the protein(s) of interest. Thirty‐one prokaryotic, eukaryotic, and viral membrane proteins were analyzed by our small‐scale procedure to identify the best‐associated detergents. Examples of results obtained with a bitopic and multitopic membrane proteins and membrane protein complexes are presented in more detail. DDM, DM, DMNG, TritonX‐100, LAPAO, and Fos‐12 appeared effective for successful membrane solubilization and protein purification of most selected targets. Eukaryotic proteins are in general more difficult to extract and purify from Escherichia coli membranes than prokaryotic proteins. The protocol has been developed for His‐tagged proteins, but can readily be adapted to other affinity tags by adjusting the chromatography resin and the buffer composition.

Rapid screening and scale‐up of ultracentrifugation‐free, membrane‐based procedures for purification of His‐tagged membrane proteins

“…In an earlier study, we hypothesized that higher micelle hydrophobicities promote partitioning of the hydrophobic MPs into the micelles . The detergent micelle hydrophobicity was determined by a fluorescence‐based assay from our previous work .…”

“…12,18 Several studies developed ultracentrifugation-free, high-throughput screening of soluble protein or MP purification using a series of 96-well plates for filtration, equilibration with an affinity resin, and subsequent collection of the resin, filtration, rinsing, and elution. [19][20][21] However, the use of affinity membranes combines filtration and capture steps to decrease the number of plates required as well as the number of transfer steps. In addition, the membrane-based procedure does not require the steps of washing and adding resin to wells before initiating binding.…”

An Overview of the Top Ten Detergents Used for Membrane Protein Crystallization

“…The screening can be optimized via the introduction of GFP-fusion for a rapid assessment of detergent solubilization efficiency [86] and protein stability in a certain detergent [86,87]. In cases where the high throughput is desirable, other protocols such as BMSS (Biotinylated Membranes Solubilization & Separation) [88] or fully-automated detergent screening [89] can be applied for fast-screening of 96 different detergents at once.…”

Smoothing membrane protein structure determination by initial upstream stage improvements

“…Membrane proteins (MP) constitute 20-30% of all proteins encoded by the genome of various organisms (Lantez et al 2015) and perform a wide range of essential biological functions, thus representing the largest class of protein drug targets (Bernaudat et al 2011). However and despite their biological relevance, most of these targets still do not have any assigned function (Bernaudat et al 2011), as reflected by the relatively low number of MP structures recorded in Stephen White's laboratory database (http://blanco.biomol.uci.edu/mpstruc/) -876 unique MP structures in March 2019.…”

“…However and despite their biological relevance, most of these targets still do not have any assigned function (Bernaudat et al 2011), as reflected by the relatively low number of MP structures recorded in Stephen White's laboratory database (http://blanco.biomol.uci.edu/mpstruc/) -876 unique MP structures in March 2019. Indeed, determining the structure of a MP is quite complex, mostly due to problems arising from MP low natural abundance, their toxicity when overexpressed in heterologous systems, and difficulties in purifying stable functional proteins and obtaining well-diffracting crystals (Gul et al 2014;Lantez et al 2015). To cope with MP low natural abundance that limits subsequent structural and functional studies, four different approaches have been proposed (Popot 2018), namely (1) overexpression in vivo and in situ; (2) overexpression in vivo in inclusion bodies; (3) cell-free expression (CFE) in vitro; (4) chemical synthesis for short MP or MP fragments.…”