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.

An engineered thermal-shift screen reveals specific lipid preferences of eukaryotic and prokaryotic membrane proteins

“…One of the most widely used approaches for screening homologs and optimizing membrane proteins for structural investigation is to couple their production with a C-terminal GFP tag 42 , 43 The cleavable GFP-tag enables a fast readout of expression levels 42 , 43 integrity by in-gel fluorescence 43 and monodispersity of the protein–detergent complex by FSEC 44 . GFP-based membrane protein screening platforms have been well established in E. coli 45 yeast 46 , 47 , insect 48 , and mammalian cells 49 . Thus, the extension of the GFP-based methodology should make the screening for specific lipids or other membrane protein ligands to in a high-throughput format easily applicable from a variety of expression hosts, and further aid functional and structural determination, as demonstrated here for the Na + /H + exchanger NhaA and the eukaryotic CMP–sialic acid transporter.…”

Baculovirus expression: old dog, new tricks

“…The use of adapted affinity tags is often the only means to obtain pure protein. 24,25 Numerous pipelines for expression screening and protein production at medium to large scale (250 ml-6L) have been established. 15,26,27 Preparation of multiprotein complexes in particular for structural biology applications has its own challenges and specifics.…”

Fluorophore Absorption Size Exclusion Chromatography (FA-SEC): An Alternative Method for High-Throughput Detergent Screening of Membrane Proteins

“…GFP-based expression screening and FSEC have been widely applied in prokaryotic and eukaryotic expression systems for heterologous membrane protein production, including E . coli [ 4 ], Saccharomyces cerevisiae [ 5 , 6 ], Pichia pastoris [ 7 , 8 ], insect cells [ 9 , 10 ] and human cells [ 4 ]. In recent years, modified strategies have been developed on the basis of FSEC for various specific experimental conditions.…”

“…In recent years, modified strategies have been developed on the basis of FSEC for various specific experimental conditions. For example, Hu et al [ 10 ] published a high-throughput screening method describing the protocol of expression and stability screening for eukaryotic membrane proteins using a pTriEx-based vector containing promoter components for E . coli , insect cells and mammalian cells allowing multi-host screen.…”

“…Conventionally, FSEC is performed on an HPLC system directly coupled to a fluorescence detector [ 4 ]. In some laboratories, the protein samples are solubilized in different detergents and loaded via an autosampler into a UHPLC system, with FSEC data collection over night [ 10 ]. Alternatively, FSEC profiles can also be traced by plotting the fluorescence intensities in each SEC-fractionized wells using a 96-well microplate spectrofluorometer against the fraction numbers [ 5 , 6 ].…”