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Engineering and characterization of a superfolder green fluorescent protein

A Corrigendum to this article was published on 01 September 2006

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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.

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Figure 1: Schematic representation of the GFP scaffolding.
Figure 2: GFP refolding kinetics and equilibrium renaturation plots.
Figure 3: Tolerance of folding reporter GFP and superfolder GFP to random mutation.
Figure 4: Fluorescence, expression level, and solubility of GFP fusions to eighteen P. aerophilum control proteins.
Figure 5: Three-dimensional structure of folding reporter GFP and superfolder GFP.

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Acknowledgements

The authors wish to acknowledge Hong Cai for help in collecting flow cytometry data, Brian Mark for helpful comments, and the NIH and LDRD-DR for generous support.

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Correspondence to Geoffrey S Waldo.

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Superfolder GFP is the subject of a published US patent application by Los Alamos National Laboratories on behalf of the University of California.

Supplementary information

Supplementary Fig. 1

(a) Fluorescence excitation and emission spectra for folding reporter GFP (dotted line) and superfolder GFP (solid line). (b) Ultraviolet-visible absorption spectra for folding reporter GFP (dotted line) and superfolder GFP (solid line). (c) Fluorescence photobleaching traces for indicated samples, exposed to continuous illumination in a FL600 Microplate Fluorescence Reader (Bio-Tek, Winooski, VT) (488 nm excitation, 530 nm emission, 10 nm band pass) (PDF 441 kb)

Supplementary Fig. 2

E. coli colonies on nitrocellulose membranes resting on LB agar plates, expressing indicated fluorescent protein variants as C-terminal fusions with poorly-folded ferritin (PDF 127 kb)

Supplementary Fig. 3

(a) Three-exponential fit to long term folding reporter GFP refolding progress curve, showing residual (data-fit) × 4. RMSD = 1005. (b) Three-exponential fit to short-term folding reporter GFP refolding progress curve, showing residual (data-fit) × 4. RMSD = 1505. (PDF 308 kb)

Supplementary Fig. 4

(a) Two-exponential fit to long term folding reporter GFP refolding progress curve, showing residual (data-fit) × 4. RMSD = 3030. (b) Two-exponential fit to short-term folding reporter GFP refolding progress curve, showing residual (data-fit) × 4. RMSD = 2500 (PDF 162 kb)

Supplementary Fig. 5

(a) Three-exponential fit to long term superfolder GFP refolding progress curve, showing residual (data-fit) × 4. RMSD = 1500. (b) Three-exponential fit to short-term superfolder GFP refolding progress curve, showing residual (data-fit) × 4. RMSD = 980 (PDF 359 kb)

Supplementary Fig. 6

(a) Two-exponential fit to long term superfolder GFP refolding progress curve, showing residual (data-fit) × 4. RMSD = 5300. (b) Two-exponential fit to short-term superfolder GFP refolding progress curve, showing residual (data-fit) × 4. RMSD = 4280 (PDF 164 kb)

Supplementary Fig. 7

(a) Fluorescence recovery for urea-denatured folding reporter GFP as a function of time after dilution to the indicated final concentrations of urea in TNG buffer. (b) Fluorescence recovery for urea-denatured superfolder GFP as a function of time after dilution to the indicated final concentrations of urea in TNG buffer (PDF 67 kb)

Supplementary Fig. 8

Interactions involving crystal contacts for folding reporter GFP (upper) and superfolder GFP (lower) (PDF 1152 kb)

Supplementary Table 1

Primers used to engineer circular permutants (PDF 70 kb)

Supplementary Table 2

Crystallographic statistics (PDF 71 kb)

Supplementary Table 3 (PDF 89 kb)

Supplementary Methods

Primers for cloning fluorescent proteins, and engineering color variants of GFP and for SF GFP mutations in the FR GFP scaffolding (PDF 18 kb)

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Pédelacq, JD., Cabantous, S., Tran, T. et al. Engineering and characterization of a superfolder green fluorescent protein. Nat Biotechnol 24, 79–88 (2006). https://doi.org/10.1038/nbt1172

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