Engineering and characterization of a superfolder green fluorescent protein

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  • A Corrigendum to this article was published on 01 September 2006

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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References

  1. 1

    Tsien, R.Y. The green fluorescent protein. Annu. Rev. Biochem. 67, 509–544 (1998).

  2. 2

    Cormack, B.P., Valdivia, R.H. & Falkow, S. FACS-optimized mutants of the green fluorescent protein (GFP). Gene 173, 33–38 (1996).

  3. 3

    Crameri, A., Whitehorn, E.A., Tate, E. & Stemmer, W.P. Improved green fluorescent protein by molecular evolution using DNA shuffling. Nat. Biotechnol. 14, 315–319 (1996).

  4. 4

    Tsukamoto, T. et al. Visualization of gene activity in living cells. Nat. Cell. Biol. 2, 871–878 (2000).

  5. 5

    Ayoob, J.C., Shaner, N.C., Sanger, J.W. & Sanger, J.M. Expression of green or red fluorescent protein (GFP or DsRed) linked proteins in non-muscle and muscle cells. Mol. Biotechnol. 17, 65–71 (2001).

  6. 6

    Babiychuk, E., Van Montagu, M. & Kushnir, S. N-terminal domains of plant poly(ADP-ribose) polymerases define their association with mitotic chromosomes. Plant J. 28, 245–255 (2001).

  7. 7

    Bachi, A. et al. The C-terminal domain of TAP interacts with the nuclear pore complex and promotes export of specific CTE-bearing RNA substrates. RNA 6, 136–158 (2000).

  8. 8

    Brumwell, C., Antolik, C., Carson, J.H. & Barbarese, E. Intracellular trafficking of hnRNP A2 in oligodendrocytes. Exp. Cell Res. 279, 310–320 (2002).

  9. 9

    Waldo, G.S., Standish, B.M., Berendzen, J. & Terwilliger, T.C. Rapid protein-folding assay using green fluorescent protein. Nat. Biotechnol. 17, 691–695 (1999).

  10. 10

    Waldo, G.S. Genetic screens and directed evolution for protein solubility. Curr Opin. Chem. Biol. 7, 33–38 (2003).

  11. 11

    Nakayama, M. & Ohara, O. A system using convertible vectors for screening soluble recombinant proteins produced in Escherichia coli from randomly fragmented cDNAs. Biochem. Biophys. Res. Commun. 312, 825–830 (2003).

  12. 12

    Pedelacq, J.D. et al. Engineering soluble proteins for structural genomics. Nat. Biotechnol. 20, 927–932 (2002).

  13. 13

    Peelle, B. et al. Intracellular protein scaffold-mediated display of random peptide libraries for phenotypic screens in mammalian cells. Chem. Biol. 8, 521–534 (2001).

  14. 14

    Baird, G.S., Zacharias, D.A. & Tsien, R.Y. Circular permutation and receptor insertion within green fluorescent proteins. Proc. Natl. Acad. Sci. USA 96, 11241–11246 (1999).

  15. 15

    Topell, S., Hennecke, J. & Glockshuber, R. Circularly permuted variants of the green fluorescent protein. FEBS Lett. 457, 283–289 (1999).

  16. 16

    Topell, S. & Glockshuber, R. Circular permutation of the green fluorescent protein. Methods Mol. Biol. 183, 31–48 (2002).

  17. 17

    Crameri, A., Whitehorn, E.A., Tate, E. & Stemmer, W.P. Improved green fluorescent protein by molecular evolution using DNA shuffling. Nat. Biotechnol. 14, 315–319 (1996).

  18. 18

    Patterson, G.H., Knobel, S.M., Sharif, W.D., Kain, S.R. & Piston, D.W. Use of the green fluorescent protein and its mutants in quantitative fluorescence microscopy. Biophys. J. 73, 2782–2790 (1997).

  19. 19

    Heim, R., Prasher, D.C. & Tsien, R.Y. Wavelength mutations and posttranslational autoxidation of green fluorescent protein. Proc. Natl. Acad. Sci. USA 91, 12501–12504 (1994).

  20. 20

    Ormö, M. et al. Crystal structure of the Aequorea victoria green fluorescent protein. Science 273, 1392–1395 (1996).

  21. 21

    Bevis, B.J. & Glick, B.S. Rapidly maturing variants of the Discosoma red fluorescent protein (DsRed). Nat. Biotechnol. 20, 83–87 (2002).

  22. 22

    Battistutta, R., Negro, A. & Zanotti, G. Crystal structure and refolding properties of the mutant F99S/M153T/V163A of the green fluorescent protein. Proteins 41, 429–437 (2000).

  23. 23

    Stepanenko, O.V. et al. Comparative studies on the structure and stability of fluorescent proteins EGFP, zFP506, mRFP1, “dimer2”, and DsRed1. Biochemistry 43, 14913–14923 (2004).

  24. 24

    Cabantous, S., Terwilliger, T.C. & Waldo, G.S. Protein tagging and detection with engineered self-assembling fragments of green fluorescent protein. Nat. Biotechnol. 23, 102–107 (2005).

  25. 25

    Phillips, G.N., Jr. Structure and dynamics of green fluorescent protein. Curr Opin Struct. Biol. 7, 821–827 (1997).

  26. 26

    Zacharias, D.A., Violin, J.D., Newton, A.C. & Tsien, R.Y. Partitioning of lipid-modified monomeric GFPs into membrane microdomains of live cells. Science 296, 913–916 (2002).

  27. 27

    Dao-pin, S. et al. Structural and genetic analysis of electrostatic and other interactions in bacteriophage T4 lysozyme. Ciba Found. Symp. 161, 52–62 (1991).

  28. 28

    Yip, K.S. et al. The structure of Pyrococcus furiosus glutamate dehydrogenase reveals a key role for ion-pair networks in maintaining enzyme stability at extreme temperatures. Structure 3, 1147–1158 (1995).

  29. 29

    Goldman, A. How to make my blood boil. Structure 3, 1277–1279 (1995).

  30. 30

    Shagin, D.A. et al. GFP-like proteins as ubiquitous metazoan superfamily: evolution of functional features and structural complexity. Mol. Biol. Evol. 21, 841–850 (2004).

  31. 31

    Magliery, T.J. & Regan, L. Combinatorial approaches to protein stability and structure. Eur. J. Biochem. 271, 1595–1608 (2004).

  32. 32

    Minor, D.L., Jr. & Kim, P.S. Measurement of the beta-sheet-forming propensities of amino acids. Nature 367, 660–663 (1994).

  33. 33

    Ewert, S., Honegger, A. & Pluckthun, A. Structure-based improvement of the biophysical properties of immunoglobulin VH domains with a generalizable approach. Biochemistry 42, 1517–1528 (2003).

  34. 34

    Fane, B. & King, J. Intragenic suppressors of folding defects in the P22 tailspike protein. Genetics 127, 263–277 (1991).

  35. 35

    Fane, B., Villafane, R., Mitraki, A. & King, J. Identification of global suppressors for temperature-sensitive folding mutations of the P22 tailspike protein. J. Biol. Chem. 266, 11640–11648 (1991).

  36. 36

    Mitraki, A., Danner, M., King, J. & Seckler, R. Temperature-sensitive mutations and second-site suppressor substitutions affect folding of the P22 tailspike protein in vitro. J. Biol. Chem. 268, 20071–20075 (1993).

  37. 37

    Mitraki, A., Fane, B., Haase-Pettingell, C., Sturtevant, J. & King, J. Global suppression of protein folding defects and inclusion body formation. Science 253, 54–58 (1991).

  38. 38

    Sideraki, V., Huang, W., Palzkill, T. & Gilbert, H.F. A secondary drug resistance mutation of TEM-1 beta-lactamase that suppresses misfolding and aggregation. Proc. Natl. Acad. Sci. USA 98, 283–288 (2001).

  39. 39

    Huang, W. & Palzkill, T. A natural polymorphism in beta-lactamase is a global suppressor. Proc. Natl. Acad. Sci. USA 94, 8801–8806 (1997).

  40. 40

    Davis, T.N. Protein localization in proteomics. Curr. Opin. Chem. Biol. 8, 49–53 (2004).

  41. 41

    Adams, S.R. et al. New biarsenical ligands and tetracysteine motifs for protein labeling in vitro and in vivo: synthesis and biological applications. J. Am. Chem. Soc. 124, 6063–6076 (2002).

  42. 42

    Martin, B.R., Giepmans, B.N., Adams, S.R. & Tsien, R.Y. Mammalian cell-based optimization of the biarsenical-binding tetracysteine motif for improved fluorescence and affinity. Nat. Biotechnol. 23, 1308–1314 (2005).

  43. 43

    Gautier, I. et al. Homo-FRET microscopy in living cells to measure monomer-dimer transition of GFT-tagged proteins. Biophys. J. 80, 3000–3008 (2001).

  44. 44

    Stemmer, W.P. DNA shuffling by random fragmentation and reassembly: in vitro recombination for molecular evolution. Proc. Natl. Acad. Sci. USA 91, 10747–10751 (1994).

  45. 45

    Tanford, C. Protein denaturation. Adv. Protein Chem. 23, 121–282 (1968).

  46. 46

    Pace, C.N. Determination and analysis of urea and guanidine hydrochloride denaturation curves. Methods Enzymol. 131, 266–280 (1986).

  47. 47

    Collaborative Computational Project, N. The CCP4 Suite: Programs for protein crystallography. Acta Crystallogr. D Biol. Crystallogr. 50, 760–763 (1994).

  48. 48

    Brunger, A.T. et al. Crystallography & NMR system: A new software suite for macromolecular structure determination. Acta Crystallogr. D Biol. Crystallogr. 54, 905–921 (1998).

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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.

Author information

Correspondence to Geoffrey S Waldo.

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Competing interests

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

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