Preparation of Fragaceatoxin C (FraC) Nanopores

Natalie Lisa Mutter, Gang Huang, Nieck Jordy van der Heide, Florian Leonardus Rudolfus Lucas, Nicole Stéphanie Galenkamp, Giovanni Maglia, Carsten Wloka*

*Corresponding author for this work

Research output: Chapter in Book/Report/Conference proceedingChapterAcademicpeer-review

Abstract

Biological nanopores are an emerging class of biosensors with high-end precision owing to their reproducible fabrication at the nanometer scale. Most notably, nanopore-based DNA sequencing applications are currently being commercialized, while nanopore-based proteomics may become a reality in the near future.Although membrane proteins often prove to be difficult to purify, we describe a straightforward protocol for the preparation of Fragaceatoxin C (FraC) nanopores, which may have applications for DNA analysis and nanopore-based proteomics. Recombinantly expressed FraC nanopores are purified via two rounds of Ni-NTA affinity chromatography before and after oligomerization on sphingomyelin-containing liposomes. Starting from a plasmid vector containing the FraC gene, our method allows the production of purified nanopores within a week. Afterward, the FraC nanopores can be stored at +4 °C for several months, or frozen.

Original languageEnglish
Title of host publicationNanopore Technology
Subtitle of host publicationMethods in Molecular Biology
EditorsM.A. Fahie
Place of PublicationNew York
PublisherHumana Press
Pages3-10
Number of pages8
ISBN (Electronic)978-1-0716-0806-7
ISBN (Print)978-1-0716-0805-0
DOIs
Publication statusPublished - 2021

Publication series

NameMethods in Molecular Biology
Volume2186
ISSN (Print)1064-3745

Keywords

  • Nanotechnology
  • Nanopore
  • Porin
  • actinoporin
  • Fragaceatoxin C
  • Fra C
  • protein purification
  • protein oligomerization
  • electrophysiology
  • artificial bilayers

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