Synthetic control of green fluorescent protein

Journal of the American Chemical Society

Volumn 131, Issue 44, 2009, Pages 15988-15989

  Kent, Kevin P ^a   Oltrogge, Luke M ^a   Boxer, Steven G ^a  

^a STANFORD UNIVERSITY   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

ALTERNATIVE METHODS; CHROMOPHORE FORMATION; FULL-LENGTH PROTEINS; GREEN FLUORESCENT PROTEIN; GUANIDINE HYDROCHLORIDE; REFOLDING; SIZE EXCLUSION; STRUCTURAL ELEMENTS; SYNTHETIC CONTROL; SYNTHETIC PEPTIDE;

AMINES; FLUORESCENCE; PEPTIDES;

CHROMOPHORES;

GREEN FLUORESCENT PROTEIN; GUANIDINE; PROTEINASE; SYNTHETIC PEPTIDE; TRYPSIN;

ALPHA HELIX; ARTICLE; BIOPHYSICS; CHROMATOPHORE; FLUORESCENCE ANALYSIS; PROTEIN EXPRESSION;

GREEN FLUORESCENT PROTEINS; PEPTIDE HYDROLASES; PROTEIN CONFORMATION; PROTEIN DENATURATION; PROTEIN RENATURATION; RECOMBINANT PROTEINS; RECOMBINATION, GENETIC; TRYPSIN;

EID: 70450189533     PISSN: 00027863     EISSN: None     Source Type: Journal    
DOI: 10.1021/ja906303f     Document Type: Article

References (18)

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5. Degradation or precipitation of truncated GFPs likely causes low yields.
6. Sakamoto, S.; Kudo, K. J. Am. Chem. Soc. 2008, 130, 9574-9582
7. Huang, Y.-M.; Bystroff, C. Biochemistry 2009, 48, 929-940
8. Despite these modifications, the E. coli expression yields of the full-length protein containing the chromophore remain high (>100 mg/L). The parent GFP (see Supporting Information for full sequences) is derived from a split GFP developed for solubility assays,2 except that position 65 is reverted back to serine, which provides an optical probe for the integrity and photochemistry of GFP17,18 (see Figure 4).
9. Richards, F. M.; Vithayathil, P. J. J. Biol. Chem. 1959, 234, 1459.
10. Sniegowski, J. A.; Phail, M. E.; Wachter, R. M. Biochem. Biophys. Res. Commun. 2005, 332, 657-663
11. Any of the 11 β-strands can be removed by circular permutation, but we use the natural C-terminal 11th strand for illustration. We used this method to remove s7; this will be reported in subsequent papers.
12. 1 is GFP:loop:s11 in this scheme.
13. Niwa, H.; Inouye, S.; Hirano, T.; Matsuno, T.; Kojima, S.; Kubota, M.; Ohashi, M.; Tsuji, F. I. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 13617-13622
14. The structures of GFP:loop:s11 and ih:loop:GFP are not yet known.
15. Kinetics of reassembly of ih and s11 with ih:loop:GFP and GFP:loop:s11, incorporation of unnatural amino acids, and rate of chromophore formation in ih will be described in subsequent work.
16. We note that native chemical ligation could be used to fuse ih to the N-terminus of ih:loop:GFP.
17. Tsien, R. Y. Annu. Rev. Biochem. 1998, 67, 509-544
18. Chattoraj, M.; King, B. A.; Bublitz, G. U.; Boxer, S. G. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 8362-8367