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A Split-GFP Gateway Cloning System for Topology Analyses of Membrane Proteins in Plants.

PLoS One.. 2017-01; 
Xie W, Nielsen ME, Pedersen C, Thordal-Christensen H. Plant Defence Genetics, Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, Frederiksberg C, Denmark.
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Abstract

To understand the function of membrane proteins, it is imperative to know their topology. For such studies, a split green fluorescent protein (GFP) method is useful. GFP is barrel-shaped, consisting of 11 β-sheets. When the first ten β-sheets (GFP1-10) and the 11th β-sheet (GFP11) are expressed from separate genes they will self-assembly and reconstitute a fluorescent GFP protein. However, this will only occur when the two domains co-localize in the same cellular compartment. We have developed an easy-to-use Gateway vector set for determining on which side of the membrane the N- and C-termini are located. Two vectors were designed for making N- and C-terminal fusions between the membrane proteins... More

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