Buy one Get one Free: GFP Proteins with Antibody Binding Properties

Everyone loves BOGOs (Buy One Get One (free)). So when I saw a recent report in PNAS from Eric Shusta’s Lab about Green Fluorescent Proteins (GFPs) with antibody like binding properties I was intrigued. As authors point out GFP based binders (GFAbs) “—could act as single step detection reagents in applications such as fluorescence based ELISAs, flow cytometry, and intracellular targeting/trafficking in live cells.” The precedent for evolving proteins with antibody like binding properties already exist-anticalins, affibodies are just few examples (See complete list in my previous posting). However, in past, inserting any binding loops to GFPs dramatically reduced the fluorescent properties of the GFPs. Superfolder GFPs, a GFP variant evolved for superior stability and improved folding, have few regions that can tolerate binding loops without taking significant hits on the fluorescence properties. The approach previous attempts took was of evolving stable GFP and then trying to insert binding domains.

The current report approached the problem from other side. They first inserted two proximal binding loops into GFP and then evolved the hybrid for high fluorescence, expression and stability. Once they had the right hybrid they then used yeast display technology to create libraries of 6×106 clones from which they selected binders against various proteins. They successfully isolated binders against: streptavidin-phycoerythrin, biotin-phycoerythrin, GAPDH(glyceraldehyde 3-phosphate dehydrogenase) and TrkB (monomeric extracellular domain of a neurotrophin receptor). The binding affinities were from 70-3.2 nanomolar – not bad!

I am guessing that this is just the beginning and we will soon see multicolored fluorescent proteins with antibody like binding properties. How successful this technology will be for ELISA like applications is open for debate. Single step detection is generally less sensitive than two step assays but niche applications will certainly benefit. Single step detection using GFAbs (or RFAbs, YFAbs etc. etc.) may benefit some POCs (point of care devices) and lateral flow immunoassays but benefit over gold or organic dye labeled antibodies will have to be demonstrated.

Hey but BOGOs are good especially in bad economy!

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