Effectiveness of dual-detergent strategy using Triton X-100 in membrane protein purification

Biochem Biophys Res Commun. 2021 Nov 12;578:122-128. doi: 10.1016/j.bbrc.2021.09.031. Epub 2021 Sep 20.

Abstract

Membrane solubilization by detergents is a critical step for successful membrane protein purification. Alkyl maltoside detergents such as DDM and DM are very expensive and are commonly used to produce most of the high-quality proteins in stable and functional form. Recently, dual-detergent strategy using inexpensive detergents for membrane solubilization step has been shown to be highly effective in purifying different classes of membrane proteins in a cost-effective manner. In this work, we have monitored the effectiveness of 'dual-detergent strategy' towards successful purification of the isolated voltage sensing domain (VSD) of KvAP and the inward rectifying K+ channel, KirBac1.1. We demonstrate that the inexpensive detergent Triton X-100 extracts the activated conformation of the KvAP-VSD well without compromising the structural integrity of the sensor, and also retains its proper structural dynamics. Importantly, the cost associated with solubilizing the KvAP sensor can be reduced by ∼2000 fold. To the best of our knowledge, our results constitute the first report characterizing the purification of KvAP voltage sensor using an inexpensive detergent. However, the dual-detergent strategy using Triton X-100 for membrane solubilization is not effective for the purification of inward rectifying K+ channel, KirBac1.1 even in presence of high salt concentration during solubilization. We propose that the dual-detergent strategy will be useful for extracting stable and functional proteins that are both DDM- and DM-extractable, but will be ineffective if the protein is only DM-extractable. The relevance of the effectiveness of dual-detergent strategy with respect to the hydrophobic thickness of proteins is discussed.

Keywords: Dual-detergent strategy; KirBac1.1; KvAP sensor; Membrane solubilization; NBD and Trp fluorescence.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Membrane / chemistry
  • Cell Membrane / metabolism
  • Detergents / chemistry*
  • Membrane Proteins / chemistry
  • Membrane Proteins / isolation & purification*
  • Membrane Proteins / metabolism
  • Octoxynol / chemistry*
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / isolation & purification*
  • Recombinant Proteins / metabolism
  • Structure-Activity Relationship
  • Surface-Active Agents / chemistry*

Substances

  • Detergents
  • Membrane Proteins
  • Recombinant Proteins
  • Surface-Active Agents
  • Octoxynol