Heat treatment of thioredoxin fusions increases the purity of α-helical transmembrane protein constructs.

Mathias Schenkel,Georg Krainer,Antoine Treff,Charles M Deber,Michael Schlierf

doi:10.1002/pro.4150

Abstract

Membrane proteins play key roles in cellular signaling and transport, represent the majority of drug targets, and are implicated in many diseases. Their relevance renders them important subjects for structural, biophysical, and functional investigations. However, obtaining membrane proteins in high purities is often challenging with conventional purification steps alone. To address this issue, we present here an approach to increase the purity of α‐helical transmembrane proteins. Our approach exploits the Thioredoxin (Trx) tag system, which is able to confer some of its favorable properties, such as high solubility and thermostability, to its fusion partners. Using Trx fusions of transmembrane helical hairpin constructs derived from the human cystic fibrosis transmembrane conductance regulator (CFTR) and a bacterial ATP synthase, we establish conditions for the successful implementation of the selective heat treatment procedure to increase sample purity. We further examine systematically its efficacy with respect to different incubation times and temperatures using quantitative gel electrophoresis. We find that minute‐timescale heat treatment of Trx‐tagged fusion constructs with temperatures ranging from 50 to 90°C increases the purity of the membrane protein samples from ~60 to 98% even after affinity purification. We show that this single‐step approach is even applicable in cases where regular selective heat purification from crude extracts, as reported for Trx fusions to soluble proteins, fails. Overall, our approach is easy to integrate into existing purification strategies and provides a facile route for increasing the purity of membrane protein constructs after purification by standard chromatography approaches.

Highlights

Membrane proteins are important actors in crossmembrane cell physiology and fulfill a variety of functions ranging from cellular signaling to energy generation and transport.[1,2] They cover about 20–50% of prokaryotic and eukaryotic genomes,[2] and constitute more than 50% of all approved drug targets.[3]
We evaluate Trx fusions of the CFTR-derived mutant helical hairpin Q220R TM3/4,24 denoted as Trx–TM3/4Q220R, and the subunit C transmembrane peptide derived from the ATP synthase of Ilyobacter tartaricus (I. tartaricus),[25] denoted as Trx–subunit C
We have demonstrated here that the purity of Trx-tagged transmembrane constructs can be considerably improved, up to ~98%, by heat treatment after initial purification steps such as affinity chromatography

Summary

Introduction

Membrane proteins are important actors in crossmembrane cell physiology and fulfill a variety of functions ranging from cellular signaling to energy generation and transport.[1,2] They cover about 20–50% of prokaryotic and eukaryotic genomes,[2] and constitute more than 50% of all approved drug targets.[3]. The production of membrane proteins typically involves overexpression of constructs in heterologous hosts[10,11] and purification by chromatography techniques such as, for example, immobilized metal affinity chromatography (IMAC).[12] Prior to chromatographic separation, the protein of interest is usually extracted from the expression host cell membrane. This is achieved with adequate solubilization agents, such as detergents, to keep the hydrophobic proteins in solution as water-soluble protein–detergent complexes in order to prevent aggregation.[7,13]

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Conclusion