eGFP as an All-in-One Tag for Purification of Membrane Proteins

Tomáš Heger, Charlott Stock, Michelle Juknaviciute Laursen, Michael Habeck, Thibaud Dieudonné*, Poul Nissen

*Corresponding author for this work

Research output: Contribution to book/anthology/report/proceedingBook chapterResearchpeer-review

2 Citations (Scopus)

Abstract

Within the last decade, cryo-electron microscopy has revolutionized our understanding of membrane proteins, but they still represent challenging targets for biochemical and structural studies. The first obstacle is often to obtain high production levels of correctly folded target protein. In these cases, the use of eGFP tags is an efficient strategy, as it allows rapid screenings of expression systems, constructs, and detergents for solubilization. Additionally, eGFP tags can now be used for affinity purification with recently developed nanobodies. Here we present a series of methods based on enhanced green fluorescent protein (eGFP) fluorescence to efficiently screen for production and stabilization of detergent-solubilized eGFP-tagged membrane proteins produced in S. cerevisiae via in-gel fluorescence SDS-PAGE and fluorescence-detection size-exclusion chromatography (FSEC). Additionally, we present a protocol describing the production of affinity resin based on eGFP-binding nanobodies produced in E. coli. We showcase the purification of human ATP7B, a copper transporting P-type ATPase, as an example of the applicability of the methods.

Original languageEnglish
Title of host publicationMethods in Molecular Biology
Number of pages16
Volume2652
Place of publicationNew York
PublisherHumana Press
Publication date2023
Pages171-186
ISBN (Print)978-1-0716-3146-1
ISBN (Electronic)978-1-0716-3147-8
DOIs
Publication statusPublished - 2023
SeriesMethods in Molecular Biology
Volume2652
ISSN1064-3745

Keywords

  • ATP7B
  • eGFP
  • FSEC
  • In-gel fluorescence
  • Membrane protein
  • Nanobody
  • Purification

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