Design and applications of a clamp for Green Fluorescent Protein with picomolar affinity

Alexander Batyuk,Simon Hansen,Daniel Bojar,Jakob C Stüber,Alexander Koch,Patrick Ernst,Andreas Plückthun

doi:10.1038/s41598-017-15711-z

Abstract

Green fluorescent protein (GFP) fusions are pervasively used to study structures and processes. Specific GFP-binders are thus of great utility for detection, immobilization or manipulation of GFP-fused molecules. We determined structures of two designed ankyrin repeat proteins (DARPins), complexed with GFP, which revealed different but overlapping epitopes. Here we show a structure-guided design strategy that, by truncation and computational reengineering, led to a stable construct where both can bind simultaneously: by linkage of the two binders, fusion constructs were obtained that “wrap around” GFP, have very high affinities of about 10–30 pM, and extremely slow off-rates. They can be natively produced in E. coli in very large amounts, and show excellent biophysical properties. Their very high stability and affinity, facile site-directed functionalization at introduced unique lysines or cysteines facilitate many applications. As examples, we present them as tight yet reversible immobilization reagents for surface plasmon resonance, as fluorescently labelled monomeric detection reagents in flow cytometry, as pull-down ligands to selectively enrich GFP fusion proteins from cell extracts, and as affinity column ligands for inexpensive large-scale protein purification. We have thus described a general design strategy to create a “clamp” from two different high-affinity repeat proteins, even if their epitopes overlap.

Highlights

Since the first demonstration that the green fluorescent protein (GFP) derived from Aequorea victoria can be used to label proteins in vivo and to directly study structures and processes in cells[1], it has become an indispensable tool in cell biological research
We demonstrate a few of them here: Green fluorescent protein (GFP)-clamps were used to immobilize target proteins on surface plasmon resonance (SPR) chips, an inexpensive GFP affinity column was produced which can be used to purify GFP-tagged proteins of interest even on a large scale; fluorescently labelled GFP-clamps were used as monovalent tight-binding detection reagents in flow cytometry; and in pull-down experiments GFP-fusions could be enriched
Structures of the unfused designed ankyrin repeat proteins (DARPins) 3G61 and 3G124nc (nc indicates the use of the optimized C-cap (Mut5)33,34) in complex with enhanced GFP35,36 have been determined (PDB IDs: 5MA6, 5MA8 and 5MAK) and confirmed the epitopes (Fig. 1)

Summary

Introduction

Since the first demonstration that the green fluorescent protein (GFP) derived from Aequorea victoria can be used to label proteins in vivo and to directly study structures and processes in cells[1], it has become an indispensable tool in cell biological research (reviewed in refs[2,3]). Thousands of functionally tested GFP-fusion constructs in cell lines or living organisms exist To study such constructs, reagents with high specificity and affinity are of great interest. More sophisticated approaches use Förster resonance energy transfer (FRET) between two FPs at both termini of the protein of interest[19] or employ self-complementing split GFP variants[20] These approaches have been expanded to membrane proteins which are often difficult to express[21,22,23]. We demonstrate a few of them here: GFP-clamps were used to immobilize target proteins on surface plasmon resonance (SPR) chips, an inexpensive GFP affinity column was produced which can be used to purify GFP-tagged proteins of interest even on a large scale; fluorescently labelled GFP-clamps were used as monovalent tight-binding detection reagents in flow cytometry; and in pull-down experiments GFP-fusions could be enriched

Methods

Results

Conclusion