Concentrating membrane proteins using ultrafiltration without concentrating detergents

Abstract

Membrane proteins (MPs) are of rapidly growing interest in the design of pharmaceutical products, novel sensors, and synthetic membranes. Ultrafiltration (UF) using commercially available centrifugal concentrators is typically employed for laboratory-scale concentration of low-yield MPs, but its use is accompanied by a concomitant increase in concentration of detergent micelles. We present a detailed analysis of the hydrodynamic processes that control detergent passage during ultrafiltration of MPs and propose methods to optimize detergent passage during protein concentration in larger-scale membrane processes. Experiments were conducted using nonionic detergents, octyl-β-D glucoside (OG), and decyl-β-D maltoside (DM) with the bacterial water channel protein, Aquaporin Z (AqpZ) and the light driven chloride pump, halorhodopsin (HR), respectively. The observed sieving coefficient (So ), a measure of detergent passage, was evaluated in both stirred cell and centrifugal systems. So for DM and OG increased with increasing filtrate flux and decreasing shear rates in the stirred cell, that is, with increasing concentration polarization (CP). Similar effects were observed during filtration of MP-detergent (MPD) micelles. However, lower transmission was observed in the centrifugal system for both detergent and MPD systems. This is attributed to free convection-induced shear and hence reduced CP along the membrane surface during centrifugal UF. Thus to concentrate MPs without retention of detergent, design of UF systems that promote CP is required. Biotechnol. Bioeng. 2016;113: 2122-2130. © 2016 Wiley Periodicals, Inc.

Microfiltration of enzyme treated egg whites for accelerated detection of viable Salmonella

“…Because many of micro‐ or ultra‐filtration processes in the literature target separations involving biofunctional molecules from proteinaceous environments, maintaining the original characteristics of the target protein in these cases is crucial. Therefore, relatively mild conditions consisting of dipolar or hydrolphilic membranes, detergents, or back pressure have been used to mitigate membrane fouling while preserving the characteristics of the target proteins . Our work employed a unique approach for preventing fouling in a special situation, i.e., concentrating living microorganisms in the presence of enzyme hydrolyzed egg whites where the target is the microorganism rather than a protein.…”

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

“…Additionally, we conducted a conventional purification of KR2 using ultracentrifugation‐based separation of membrane fragments and resin‐based affinity chromatography. While we conducted overnight solubilization of KR2 for the process to be representative for rhodopsins, the solubilization duration for MPs tend to vary from as low as a few hr to overnight (∼12 hr) as shown in Figure . The resin‐based purification yielded 2.9 g KR2 per liter of cell culture which is 4.3 times the yield of ∼0.68 g KR2 per liter of cell culture with 96‐well‐based membrane purification.…”

“…Addition of a hydrophobic chelator enhances the process through phase‐separation of micelles. For both the screening and the scale‐up, we reduced the time to purify MPs from 7 to 18 hr in conventional procedures to about 4.5 hr, if not less by, for example, reducing the duration of centrifugation steps. The screening requires minimal quantity of cell culture and other consumables, and avoids the use of expensive ultracentrifuges.…”

“…Because the stability of the final protein in most extraction/purification processes depends on the detergents and buffer components used, purification of new MPs involves screening for optimal conditions to maximize both the stability and yield of protein. Typical lab‐scale preparations of MPs employ several steps including cell lysis, ultracentrifugation‐based separation of membrane fragments from cell debris, detergent‐based solubilization of MPs from the lipid bilayer, a second ultracentrifugation‐based separation of soluble or detergent‐solubilized protein from membrane fragments, and finally resin‐based affinity chromatography …”

“…Typical lab-scale preparations of MPs employ several steps including cell lysis, ultracentrifugationbased separation of membrane fragments from cell debris, detergentbased solubilization of MPs from the lipid bilayer, a second ultracentrifugation-based separation of soluble or detergentsolubilized protein from membrane fragments, and finally resin-based affinity chromatography. 8,[10][11][12] Unlike conventional protein purification that utilizes resins, we have developed a membrane-based screening and purification procedure, which saves on both time and equipment requirements. The relatively large (1.2 μm) pores in these microporous membranes allow direct transfer of cell lysate containing the solubilized MPs onto the membrane for affinity binding without requiring ultracentrifugation-based removal of membrane fragments.…”