A Novel Tandem-tag Purification Strategy for Challenging Disordered Proteins

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Abstract

Intrinsically disordered proteins (IDPs) lack well-defined 3D structures and can only be described as ensembles of different conformations. This high degree of flexibility allows them to interact promiscuously and makes them capable of fulfilling unique and versatile regulatory roles in cellular processes. These functional benefits make IDPs widespread in nature, existing in every living organism from bacteria and fungi to plants and animals. Due to their open and exposed structural state, IDPs are much more prone to proteolytic degradation than their globular counterparts. Therefore, the purification of recombinant IDPs requires extra care and caution, such as maintaining low temperature throughout the purification, the use of protease inhibitor cocktails and fast workflow. Even so, in the case of long IDP targets, the appearance of truncated by-products often seems unavoidable. The separation of these unwanted proteins can be very challenging due to their similarity to the parent target protein. Here, we describe a tandem-tag purification method that offers a remedy to this problem. It contains only common affinity-chromatography steps (HisTrap and Heparin) to ensure low cost, easy access and scaling-up for possible industrial use. The effectiveness of the method is demonstrated with four examples, Tau-441 and two of its fragments and the transactivation domain (AF1) of androgen receptor.
Original languageEnglish
Article number1566
Pages (from-to)1-19
Number of pages19
JournalBiomolecules
Volume12
Issue number11
DOIs
Publication statusPublished - Nov 2022

Bibliographical note

Funding Information:
This work was supported by a VUB Strategic Research Program on Microfluidics (SRP51) at Vrije Universiteit Brussel (VUB, Brussels, Belgium, to P.T.), an EC H2020-WIDESPREAD-2020-5 Twinning grant (PhaseAGE, no. 952334, to P.T.), an EC H2020-MSCA-RISE Action grant (IDPfun, no. 778247, to P.T.), grants K124670 and K131702 from the National Research, Development and Innovation Office (NKFIH, Hungary, to P.T.) and FWO PhD fellowships in strategic basic research (FWOSB77, to J.A.) and fundamental research (11D0122N-BIO1, to K.M.).

Publisher Copyright:
© 2022 by the authors.

Copyright:
Copyright 2022 Elsevier B.V., All rights reserved.

Keywords

  • Intrinsically disordered proteins (IDPs)
  • Protein purification
  • Affinity chromatography
  • Tau
  • Androgen receptor

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