Structural Proteomics pp 197-209

Part of the Methods in Molecular Biology book series (MIMB, volume 1261)

| Cite as

GFP-Based Expression Screening of Membrane Proteins in Insect Cells Using the Baculovirus System

  • Nien-Jen Hu
  • Heather Rada
  • Nahid Rahman
  • Joanne E. Nettleship
  • Louise Bird
  • So Iwata
  • David Drew
  • Alexander D. Cameron
  • Raymond J. Owens
Protocol

Abstract

A key step in the production of recombinant membrane proteins for structural studies is the optimization of protein yield and quality. One commonly used approach is to fuse the protein to green fluorescent protein (GFP), enabling expression to be tracked without the need to purify the protein. Combining fusion to green fluorescent protein with the baculovirus expression system provides a useful platform for both screening and production of eukaryotic membrane proteins. In this chapter we describe our protocol for the expression screening of membrane proteins in insect cells using fusion to GFP as a reporter. We use both SDS-PAGE with in-gel fluorescence imaging and fluorescence-detection size-exclusion chromatography (FSEC) to screen for expression.

Key words

Membrane proteins Baculovirus Insect cells Green fluorescent protein Fluorescence-detection size-exclusion chromatography 

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Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Nien-Jen Hu
    • 2
  • Heather Rada
    • 3
  • Nahid Rahman
    • 3
  • Joanne E. Nettleship
    • 3
  • Louise Bird
    • 3
  • So Iwata
    • 1
  • David Drew
    • 5
  • Alexander D. Cameron
    • 6
  • Raymond J. Owens
    • 3
  1. 1.Division of Molecular BiosciencesImperial College LondonLondonUK
  2. 2.Institute of BiochemistryNational Chung Hsing UniversityTaichungTaiwan
  3. 3.Oxford Protein Production Facility-UK, Research Complex at Harwell, R92 Rutherford Appleton LaboratoriesHarwell OxfordOxfordUK
  4. 4.Division of Structural BiologyWellcome Trust Centre for Human Genetics Roosevelt DriveOxfordUK
  5. 5.Research Complex at HarwellDepartment of Biochemistry and Biophysics, Stockholm UniversityStockholmSweden
  6. 6.School of Life SciencesUniversity of WarwickCoventryUK

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