Engineering and characterization of a superfolder green fluorescent protein

Abstract

Existing variants of green fluorescent protein (GFP) often misfold when expressed as fusions with other proteins. We have generated a robustly folded version of GFP, called 'superfolder' GFP, that folds well even when fused to poorly folded polypeptides. Compared to 'folding reporter' GFP, a folding-enhanced GFP containing the 'cycle-3' mutations and the 'enhanced GFP' mutations F64L and S65T, superfolder GFP shows improved tolerance of circular permutation, greater resistance to chemical denaturants and improved folding kinetics. The fluorescence of Escherichia coli cells expressing each of eighteen proteins from Pyrobaculum aerophilum as fusions with superfolder GFP was proportional to total protein expression. In contrast, fluorescence of folding reporter GFP fusion proteins was strongly correlated with the productive folding yield of the passenger protein. X-ray crystallographic structural analyses helped explain the enhanced folding of superfolder GFP relative to folding reporter GFP.

Apical and lateral cell protrusions interconnect epithelial cells in live Drosophila wing imaginal discs

“…To test whether Promininlike localizes to apical protrusions, we fused Drosophila Prominin-like to GFP and expressed the fusion protein using the GAL4/UAS system in a subset of columnar wing imaginal disc cells. By analyzing live wing imaginal discs expressing the Prominin-like-GFP fusion protein, we noticed, however, that the GFP fluorescence was undetectable (data not shown), possibly because the proper folding of the GFP molecule was impaired in the context of the fusion protein, as described for other GFP-fusion proteins (Pedelacq et al, 2006;and references therein). We therefore tested whether Prominin-like-GFP localized to apical protrusions of wing imaginal disc cells by immunostaining of fixed wing imaginal discs.…”

SAXS/SANS on Supercharged Proteins Reveals Residue-Specific Modifications of the Hydration Shell

“…S1) were designed based on super-charged GFPs (54). All three were synthesized and cloned into pET30a(þ) vectors (GeneCust), expressed as His-GFP fusion proteins in Escherichia coli BL21(DE3), and purified according to previously established protocols (52,54), with minor modifications. Full details are provided in the Supporting Material.…”

“…All GFP protein constructs used in this study contained flexible regions of 14 residues (10 at the C-termini and 4 at the N-termini) that are not resolved in the homologous GFP crystal structure (52). We therefore adopted an ensemble generation algorithm, flexible-meccano (53,59), to account for them by creating a pool of conformers with different tail conformations that were fitted to the experimental SAXS data using CRYSOL (35).…”

Approaches to imaging unfolded secretory protein stress in living cells

“…At this time, we recommend superfolder GFP [56, 57], secBFP2 [47], and FusionRed [58] for protein fusions. For transcriptional reporters, mNeonGreen matures rapidly and produces an intense signal [59] and TagRFP has similar properties for a red reporter [60].…”