title: GFP-based expression screening of membrane proteins in insect cells using the baculovirus system.
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.
authors.affiliation: Institute of Biochemistry, National Chung Hsing University, Taichung, 40227, Taiwan, njhu@nchu.edu.tw.
authors.forename: Nien-Jen
authors.initials: NJ
authors.lastname: Hu
authors.forename: Heather
authors.initials: H
authors.lastname: Rada
authors.forename: Nahid
authors.initials: N
authors.lastname: Rahman
authors.forename: Joanne E
authors.initials: JE
authors.lastname: Nettleship
authors.forename: Louise
authors.initials: L
authors.lastname: Bird
authors.forename: So
authors.initials: S
authors.lastname: Iwata
authors.forename: David
authors.initials: D
authors.lastname: Drew
authors.forename: Alexander D
authors.initials: AD
authors.lastname: Cameron
authors.forename: Raymond J
authors.initials: RJ
authors.lastname: Owens
mesh_terms: D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D005822:Genetic Vectors / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins / Q000096:biosynthesis*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D061987:Sf9 Cells; D018411:Spodoptera / Q000166:cytology / Q000821:virology*
chemical_list: D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins