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

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Schenkel, Mathias 
Treff, Antoine 
Deber, Charles M. 


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.


Funder: Boehringer Ingelheim Stiftung; Id:

Funder: Cystic Fibrosis Canada; Id:

Funder: European Social Fund; Id:

Funder: Herchel Smith Funds

Funder: Mukoviszidose Institut gGmbH

Funder: Wolfson College Junior Research Fellowship


Methods and Applications, heat treatment, membrane proteins, protein purification, thioredoxin

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Protein Science

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John Wiley & Sons, Inc.
Canadian Institutes of Health Research (376666)
H2020 Marie Skłodowska‐Curie Actions (841466)