CaltechAUTHORS
  A Caltech Library Service

Evolution of a fluorinated green fluorescent protein

Yoo, Tae-Hyeon and Link, A. James and Tirrell, David A. (2007) Evolution of a fluorinated green fluorescent protein. Proceedings of the National Academy of Sciences of the United States of America, 104 (35). pp. 13887-13890. ISSN 0027-8424. PMCID PMC1955812. https://resolver.caltech.edu/CaltechAUTHORS:YOOpnas07

[img]
Preview
PDF - Published Version
See Usage Policy.

543Kb
[img]
Preview
PDF - Supplemental Material
See Usage Policy.

18Kb
[img]
Preview
PDF (Fig. 4. Schematic representation of the GFPm scaffold. Two mutations (S65G and S72A, red circles) were introduced into the cycle-3 GFP mutant (Q80R, F99S, M153T, and V163A, red circles) to change the spectral properties; 19 Leu residues (blue circles)) - Supplemental Material
See Usage Policy.

142Kb
[img]
Preview
PDF (Fig. 5. MALDI-MS analysis of GFPm-T after trypsin digestion. A peptide fragment of sequence GEELFTGVVPILVELDGDVNGHK yields the spectrum shown. Replacement of Leu with Tfl results in a 54-amu mass increase per site.) - Supplemental Material
See Usage Policy.

86Kb
[img]
Preview
PDF (Fig. 6. Flow cytometric analysis of cells expressing the first library in media supplemented with 1 mM Tfl and no Leu (A), 15 mM Leu (B), 30 mM Leu (C), or 45 mM Leu (D).) - Supplemental Material
See Usage Policy.

119Kb
[img]
Preview
PDF (Fig. 7. Flow cytometric analysis of cells expressing proteins in media supplemented with 1 mM Tfl and 45 mM Leu. (A) Cells harboring the first library. (B) Cells after two rounds of sorting starting from A.) - Supplemental Material
See Usage Policy.

100Kb
[img]
Preview
PDF (Fig. 8. Fluorescence spectra of 11.3.3-L before (black) and after (blue) acid denaturation. Purified protein was unfolded in 100 mM citrate (pH 1.9) containing 2 mM DTT at room temperature for 1 h....) - Supplemental Material
See Usage Policy.

85Kb
[img]
Preview
PDF (Fig. 9. Equilibrium acid denaturation plots. The purified proteins were equilibrated with 100 mM buffer (Hepes for pH 8, 7.5, and 7; Mes for pH 6.5, 6, and 5.5; and acetate for pH 5, 4.5, 4) containing 100 mM NaCl at room temperature....) - Supplemental Material
See Usage Policy.

108Kb
[img]
Preview
PDF (Fig. 10. Effects of mutation at position 64 of 11.3.3. ...) - Supplemental Material
See Usage Policy.

77Kb
[img]
Preview
PDF (Fig. 11. Overlay of the normalized g(s*) plots from DcD+ analysis for GFPm-L(A), 11.3.3-L (B), and 11.3.3-T (C). Black line: 0.125 mg/ml, blue line: 0.25 mg/ml, pale green line: 0.5 mg/ml) - Supplemental Material
See Usage Policy.

159Kb

Use this Persistent URL to link to this item: https://resolver.caltech.edu/CaltechAUTHORS:YOOpnas07

Abstract

The fluorescence of bacterial cells expressing a variant (GFPm) of the green fluorescent protein (GFP) was reduced to background levels by global replacement of the leucine residues of GFPm by 5,5,5-trifluoroleucine. Eleven rounds of random mutagenesis and screening via fluorescence-activated cell sorting yielded a GFP mutant containing 20 amino acid substitutions. The mutant protein in fluorinated form showed improved folding efficiency both in vivo and in vitro, and the median fluorescence of cells expressing the fluorinated protein was improved {approx}650-fold in comparison to that of cells expressing fluorinated GFPm. The success of this approach demonstrates the feasibility of engineering functional proteins containing many copies of abiological amino acid constituents.


Item Type:Article
Related URLs:
URLURL TypeDescription
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1955812/PubMed CentralArticle
http://www.pnas.org/cgi/content/full/0701904104/DC1PublisherSupporting Information
ORCID:
AuthorORCID
Tirrell, David A.0000-0003-3175-4596
Additional Information:© 2007 by the National Academy of Sciences. Edited by David H. Baker, University of Illinois at Urbana–Champaign, Urbana, IL, and approved July 17, 2007 (received for review March 5, 2007). Published online on August 23, 2007, 10.1073/pnas.0701904104 We thank Jin K. Montclare and Frances H. Arnold for helpful discussion. We also thank Mona Shahgholi for assistance with MALDI-MS. This work was supported by National Institutes of Health Grant GM62523 and Office of Naval Research Grant N00014-03-1-0793 (to D.A.T) and by a Samsung Scholarship (to T.H.Y.). Author contributions: T.H.Y. and D.A.T. designed research; T.H.Y. performed research; T.H.Y. and D.A.T. analyzed data; and T.H.Y., A.J.L., and D.A.T. wrote the paper. The authors declare no conflict of interest. This article is a PNAS Direct Submission. This article contains supporting information online at www.pnas.org/cgi/content/full/0701904104/DC1.
Funders:
Funding AgencyGrant Number
NIHGM62523
Office of Naval Research (ONR)N00014-03-1-0793
Samsung ScholarshipUNSPECIFIED
Subject Keywords:directed evolution; noncanonical amino acids; protein engineering
Issue or Number:35
PubMed Central ID:PMC1955812
Record Number:CaltechAUTHORS:YOOpnas07
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:YOOpnas07
Alternative URL:http://dx.doi.org/10.1073/pnas.0701904104
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:9676
Collection:CaltechAUTHORS
Deposited By: Archive Administrator
Deposited On:29 Feb 2008
Last Modified:03 Oct 2019 00:02

Repository Staff Only: item control page