Journal of Molecular Biology
Expansion of the Genetic Code Enables Design of a Novel “Gold” Class of Green Fluorescent Proteins
Introduction
Green fluorescent protein (GFP) from the jellyfish Aequoria victoria (av) has become a standard reporter in cellular and molecular biology.1 Its chromophore (4-(p-hydroxybenzylidene)imidazolidin-5-one) is encoded in the amino acid sequence and autocatalytically formed in the post-translational reaction between the side-chains of the residues 65–57.2., 3. The presence of an aromatic amino acid at position 66 is crucial for the formation of fluorescent GFPs.4 The standard genetic code, however limits the number of possible amino acids at this position to the canonical residues Trp, Tyr, His and Phe. It is therefore not surprising that despite years of efforts using classical protein engineering approaches, no significantly red-shifted variants of avFP were found (i.e. not emitting beyond 530 nm).5
The most straightforward way to break these limits is the replacement of some canonical (i.e. coded) aromatic amino acids with non-canonical (i.e. non-coded) ones in template-directed protein synthesis. This can be achieved through the use of amino acid analogues, which mimic their natural counterparts.6 We and others recently succeeded in accommodating an additional Trp-like amino acid or its surrogate as a response to UGG Trp codon-containing DNA templates into proteins.7 The list of these novel translationally active amino acids include fluorinated analogues like (4-F)Trp, (5-F)Trp, (6-F)Trp, aza-analogues ((7-aza)Trp), hydroxy-((4-OH)Trp or (5-OH)Trp), and amino-Trp-analogues ((4-NH2)Trp and (5-NH2)Trp) and even Trp-surrogates such as β-(thienopyrrolyl)alanines or β-(selenolopyrrolyl)alanines. In GFP, the substitution of chromophore building residues is immediately affecting its optical properties. GFP therefore is an ideal model for the observation of such insertions.
The promise of this method is exemplified by the introduction of an electron-donating amino group in tryptophan of “enhanced cyan fluorescent protein” ECFP.4 The resulting “gold” fluorescent protein, GdFP, is the most red-shifted avFP-variant known to date (69 nm when compared to the parent ECFP and 47 nm when compared to the “enhanced yellow fluorescent protein”, EYFP (Figure 1). GdFP not only has a red-shifted emission, but also an increased thermostability, and a decreased tendency to aggregate. It is expressed and purified using Trp-auxotrophic Escherichia coli strain ATCC 499807 in quantities comparable to parent proteins (≈30 mg per litre of the culture). This provided enough material for successful crystallisation, structure elucidation, and various biochemical and biophysical characterisations.
Section snippets
Breaking the limits of classical GFP engineering
The approach for in vivo incorporation of non-canonical amino acids into proteins used here, is based on the use of auxotrophic E. coli host strains for selective pressure incorporation (SPI).8., 9. It relies on the relaxed substrate specificity of aminoacyl-tRNA synthetases as the crucial enzymes in the interpretation of the genetic code. Thus, the activation and transfer onto cognate tRNAs of a variety of structurally and chemically similar substrate analogues is possible. The SPI method does
Chemicals, analogue incorporation, fermentation and protein purification
The amino acids l-Trp, (4-Me)-Trp, and (6-F)-Trp were purchased from Bachem, while amino and hydroxy-derivatives of Trp were synthesised as described elsewhere.7 Recombinant wt, and labelled protein expression experiments were performed in E. coli ATTC499807., 23., 24. transformed with a pQE80 (Qiagen) harbouring Nt-His-Tagged EGFP and ECFP gene sequence (BD Biosciences Clontech). The cells were first grown in minimal medium in the presence of 0.015 mM l-Trp as natural substrate until the Trp
Supplementary Files
Acknowledgements
We thank Mrs E. Weyher for mass-spectrometric measurements, Ms T. Krywcun, Ms P. Birle for help in cloning work, and Mrs W. Wenger and our practical student A. Siegl for help in preparative work. G. J. and A. Z. thank R. Bausinger for help with the confocal microscopy and C. Bräuchle for continuous support. M. K. A. is the recipient of a Humboldt Fellowship. Financial support was provided by the SFB 533 and by the BMBF-Projekt BIOC8054.
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