green-fluorescent-protein-based-expression-screening-membrane

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in <em>Escherichia coli</em>

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

Research Complex at Harwell

In-Fusion™ cloning is a flexible DNA ligase-independent cloning technology that has wide-ranging uses in molecular biology. In this chapter we describe the protocols used in the OPPF-UK to design and construct expression vectors using In-Fusion™. Our method for small scale expression screening in Escherichia coli of constructs generated by In-Fusion™ is also outlined.

Application of In-Fusion™ cloning for the parallel construction of E. coli expression vectors.

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.

Article	Year
Application of In-Fusion™ cloning for the parallel construction of E. coli expression vectors. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 24395367	2014
GFP-Based Expression Screening of Membrane Proteins in Insect Cells Using the Baculovirus System. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 25502201	2015

Heather Rada

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