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

Abstract

This study develops procedures to rapidly screen conditions for purification of membrane proteins (MPs) using 96‐well plates containing nickel‐functionalized membranes. In addition to their application in the pharmaceutical industry, MPs are important components of new sensors, synthetic membranes, and bioelectronic devices. However, purification of MPs is challenging due to their hydrophobic exterior, which requires stabilization in amphipathic detergent micelles. We examined the extent of extraction of the light‐driven sodium transporter, Krokinobacter eikastus rhodopsin 2 (KR2) heterologously expressed in Escherichia coli using different salts and maltoside‐based detergents. The extraction was followed by subsequent affinity purification in membranes functionalized with Ni2+‐nitrilotriacetate complexes that bind the His‐tagged KR2. We also employed a hydrophobic chelator to separate detergent micelles from the aqueous phase as an initial isolation step prior to affinity purification. Unlike conventional resin‐based capture, which can take a full day or more, the membrane‐based screening of purification conditions takes only a few hours, and its scale‐up involves changing from a 96‐well format to a larger membrane module. The novelty of the method lies in utilizing membrane‐based ultracentrifugation‐free purification of MPs from cell membrane fragments; the optimized purification conditions from the screening method can potentially be applied to large‐scale/conventional resin‐based purification of MPs.

Calcium‐Mediated Liposome Fusion to Engineer Giant Lipid Vesicles with Cytosolic Proteins and Reconstituted Mammalian Proteins

“…To generate these proteincarrying small or large unilamellar vesicles (SUVs or LUVs), membrane proteins are extracted and purified from host cells overexpressing the protein of interest. [5][6][7] The purified proteins are solubilized in amphiphilic agents, for example, in detergent micelles. Mixing the solubilized proteins with lipids, followed by a detergent removal step, yields submicron proteo-liposomes.…”

Surrogate model to screen for inactivation‐based clearance of enveloped viruses during biotherapeutics process development

“…Detergents tested include OG (Octyl glucoside), DDM (Dodecyl-β-D-maltopyranoside), NaTC (Sodium taurocholate), and Trehalose-6-dodecanoate. [31][32][33][34][35][36] Known VI agents Triton X-100, Ecosurf [8] and LDAO (Lauryldimethylamineoxide) [9] have been tested as positive controls. A comprehensive list of detergent can be found in the supplementary information (SI), Table S1 and a schematic of the phage inactivation and quantification methods are shown in Figure 1.…”

Recent Developments in Bioprocessing of Recombinant Proteins: Expression Hosts and Process Development

“…A 10-fold decrease in buffer, resin, and time of purification was observed in comparison to conventional columns for similar protein yields. In a recent study, using 96well-plates containing nickel-functionalized membranes, rapid screening of parameters for membrane protein purification was successfully performed (Feroz et al, 2019). Mixed-mode resins (ionic and hydrophobic interactions) were used in a plate-based HT screening platform for the selection of process parameters to achieve high purity and high overall yield of osteopontin (Guo et al, 2019).…”