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

Hasin Feroz, Joshua Meisenhelter, Gia Jokhadze, Merlin Bruening, Manish Kumar

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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.

Original languageEnglish (US)
Article numbere2859
JournalBiotechnology progress
Volume35
Issue number6
DOIs
StatePublished - Nov 1 2019

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All Science Journal Classification (ASJC) codes

  • Biotechnology

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