Evolution of a fluorinated green fluorescent protein

Yoo, Tae Hyeon; Link, A. James; Tirrell, David A.

doi:10.1073/pnas.0701904104

Cited by 100 publications

(103 citation statements)

References 30 publications

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“…In contrast to previous attempts that include unnatural or noncanonical amino acids in directed evolution experiments (21,22), unnatural amino acids in our system are genetically encoded and require only the unique codon, TAG, for site-specific incorporation. Therefore, translation with unnatural amino acids utilizes the same fundamental paradigm as translation with natural amino acids, resulting in unrestricted 21 aa protein evolution.…”

Section: Discussionmentioning

confidence: 99%

Protein evolution with an expanded genetic code

Liu

Mack

Tsao

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

143

132

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We have devised a phage display system in which an expanded genetic code is available for directed evolution. This system allows selection to yield proteins containing unnatural amino acids should such sequences functionally outperform ones containing only the 20 canonical amino acids. We have optimized this system for use with several unnatural amino acids and provide a demonstration of its utility through the selection of anti-gp120 antibodies. One such phage-displayed antibody, selected from a naïve germline scFv antibody library in which six residues in V H CDR3 were randomized, contains sulfotyrosine and binds gp120 more effectively than a similarly displayed known sulfated antibody isolated from human serum. These experiments suggest that an expanded ''synthetic'' genetic code can confer a selective advantage in the directed evolution of proteins with specific properties. directed evolution ͉ phage display ͉ unnatural amino acids W ith few exceptions, the genetic codes of all known organisms specify only the 20 canonical amino acids for protein synthesis. Yet it is quite possible that the ability to encode additional amino acids and their corresponding chemical functionalities would be evolutionarily advantageous, especially since nature's choice of 20 could have been arbitrarily fixed at the point of transition between communal and Darwinian evolution paradigms and subsequently sustained by the code's inertia (1). Furthermore, in the limited scope of laboratory-directed evolution, which concerns only one or few specific functions over a short time rather than general organismal fitness over thousands of years, one can easily envision a selective advantage conferred by additional amino acids. Recent developments in our laboratory allow us to explore this possibility. Specifically, orthogonal tRNA/aminoacyl-tRNA synthetase (aaRS) pairs capable of incorporating various unnatural amino acids into proteins in response to unique nonsense and frameshift codons have been added to the translational machinery of Escherichia coli (2). These E. coli (X-E. coli) can now be used for evolution of protein function wherein 21 building blocks, rather than the common 20, are available.Several unnatural amino acids were initially chosen, on the basis of their unique chemistries, for use in our system. For example, X-E. coli genetically encoding the bidentate metalchelating amino acid bipyridyl-alanine (3) are well-suited for the evolution of redox and hydrolytic catalysts, as metal ion binding would not require preorganized primary and secondary ligand shells. Similarly, X-E. coli encoding the reactive 4-boronophenylalanine (4) are well-suited for evolution of receptors specific for glycoproteins or serine protease inhibitors, because the boronate group can form covalent complexes with diols or reactive serine residues. In addition, X-E. coli genetically encoding otherwise posttranslationally modified amino acids, such as sulfotyrosine (5), can be used for evolution of properties that exploit the unique chemical characterist...

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Section: Discussionmentioning

confidence: 99%

Protein evolution with an expanded genetic code

Liu

Mack

Tsao

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

143

132

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“…A similar approach was used for the incorporation of Tfl into a GFP mutant as mentioned above. 148 Here, the substitution of 78% of the Leu residues led to a vanishing of the protein's fluorescence as the protein was not correctly folded. After 11 rounds of evolution the incorporation efficiency could be increased to 87% and -even more importantly -the fluorescence could be restored.…”

mentioning

confidence: 99%

Organic fluorine as a polypeptide building element: in vivo expression of fluorinated peptides, proteins and proteomes

Merkel

Budiša

2012

Org. Biomol. Chem.

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“…So far, construction of artificial biological systems (1)(2)(3)(4)(5) is limited by the existing metabolic capabilities. By supplying living cells with chemically synthesized nonnatural amino acids (6,7) and sugars (8) as new building blocks, it is possible to introduce novel physical and chemical properties into biological entities. These efforts raise an interesting question: Can we rewire metabolism in a bottom-up fashion to produce nonnatural metabolites from simple carbon source?…”

mentioning

confidence: 99%

Expanding metabolism for biosynthesis of nonnatural alcohols

Zhang¹,

Sawaya

Eisenberg

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

372

264

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Nature uses a limited set of metabolites to perform all of the biochemical reactions. To increase the metabolic capabilities of biological systems, we have expanded the natural metabolic network, using a nonnatural metabolic engineering approach. The branched-chain amino acid pathways are extended to produce abiotic longer chain keto acids and alcohols by engineering the chain elongation activity of 2-isopropylmalate synthase and altering the substrate specificity of downstream enzymes through rational protein design. When introduced into Escherichia coli, this nonnatural biosynthetic pathway produces various long-chain alcohols with carbon number ranging from 5 to 8. In particular, we demonstrate the feasibility of this approach by optimizing the biosynthesis of the 6-carbon alcohol, (S)-3-methyl-1-pentanol. This work demonstrates an approach to build artificial metabolism beyond the natural metabolic network. Nonnatural metabolites such as long chain alcohols are now included in the metabolite family of living systems.metabolic engineering ͉ protein engineering ͉ chain elongation ͉ long chain alcohols N ature uses a limited set of metabolites such as organic acids, amino acids, nucleotides, lipids and sugars as building blocks for biosynthesis. These chemicals support the biological functions of all organisms. So far, construction of artificial biological systems (1-5) is limited by the existing metabolic capabilities. By supplying living cells with chemically synthesized nonnatural amino acids (6, 7) and sugars (8) as new building blocks, it is possible to introduce novel physical and chemical properties into biological entities. These efforts raise an interesting question: Can we rewire metabolism in a bottom-up fashion to produce nonnatural metabolites from simple carbon source? If so, such engineered artificial metabolism should be able to expand the chemical repertoire that living systems can use and produce. To begin to address this question, we developed a strategy to produce 7-(C7) to 9-carbon (C9) 2-keto acids, which can lead to useful nonnatural alcohols (C6-C8).Aliphatic alcohols with carbon chain length Ͼ5 (C Ͼ 5) are attractive biofuel targets because they have higher energy density, and lower water solubility [1-pentanol 23 g/L, 1-hexanol 6.2 g/L, 1-heptanol 1.2 g/L (9)] that could facilitate postproduction purification from culture medium through an aqueous/organic 2-phase separation process. The only well-characterized mechanism for aliphatic alcohol production is through the Ehrlich pathway (10), which converts branched-chain amino acids into alcohols. The carbon number (up to 5) of the alcohols derived from this type of pathway is limited by the carbon number in the branched chain amino acid pathways (11). To overcome this limitation, existing metabolic networks need to be expanded. This is a daunting task because a metabolic pathway usually involves the collective function of multiple enzymes, which have to be engineered by rational design (12) or directed evolution (13,14) to perform nonnat...

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Evolution of a fluorinated green fluorescent protein

Abstract: directed evolution ͉ noncanonical amino acids ͉ protein engineering

Cited by 100 publications

References 30 publications

Protein evolution with an expanded genetic code

Protein evolution with an expanded genetic code

Organic fluorine as a polypeptide building element: in vivo expression of fluorinated peptides, proteins and proteomes

Expanding metabolism for biosynthesis of nonnatural alcohols

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