Evolution of Proteins with Genetically Encoded “Chemical Warheads”

Liu, Chang C.; Mack, Antha V.; Brustad, Eric M.; Mills, Jeremy H.; Groff, Dan; Smider, Vaughn V.; Schultz, Peter G.

doi:10.1021/ja902985e

Cited by 72 publications

(64 citation statements)

References 6 publications

(13 reference statements)

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“…Since Schultz and collaborators achieved the first breakthrough, various UAAs, including derivatives of Lys and Tyr, have been incorporated in vivo (19,20). This technique has been widely applied for protein engineering in applications such as structural analyses, functional analyses, protein therapeutics, and functional improvement (23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33).…”

mentioning

confidence: 99%

High-Yield, Zero-Leakage Expression System with a Translational Switch Using Site-Specific Unnatural Amino Acid Incorporation

Minaba

Kato

2014

Appl Environ Microbiol

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Synthetic biologists construct complex biological circuits by combinations of various genetic parts. Many genetic parts that are orthogonal to one another and are independent of existing cellular processes would be ideal for use in synthetic biology. However, our toolbox is still limited with respect to the bacterium Escherichia coli, which is important for both research and industrial use. The site-specific incorporation of unnatural amino acids is a technique that incorporates unnatural amino acids into proteins using a modified exogenous aminoacyl-tRNA synthetase/tRNA pair that is orthogonal to any native pairs in a host and is independent from other cellular functions. Focusing on the orthogonality and independency that are suitable for the genetic parts, we designed novel AND gate and translational switches using the unnatural amino acid 3-iodo-L-tyrosine incorporation system in E. coli. A translational switch was turned on after addition of 3-iodo-L-tyrosine in the culture medium within minutes and allowed tuning of switchability and translational efficiency. As an application, we also constructed a gene expression system that produced large amounts of proteins under induction conditions and exhibited zero-leakage expression under repression conditions. Similar translational switches are expected to be applicable also for eukaryotes such as yeasts, nematodes, insects, mammalian cells, and plants.

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confidence: 99%

High-Yield, Zero-Leakage Expression System with a Translational Switch Using Site-Specific Unnatural Amino Acid Incorporation

Minaba

Kato

2014

Appl Environ Microbiol

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“…A phagemid expression system comprising bacteriophage M13 coat protein pIII and an orthogonal tRNA/synthetase pair was utilized for the incorporation of UAAs in response to the amber stop codon (TAG) [93]. Antibodies containing boronate or sulfotyrosine residues were found to outcompete natural antibodies in binding to acylic glucamine resins and the HIV coat protein gp120, respectively [96][97][98]. Although this strategy was demonstrated with an antibody, it can also be extended to enzymes where the incorporation of novel amino acid codons into the genetic code can be advantageous for the evolution of enzymes with novel or enhanced functions.…”

Section: Directed Evolution With Site-specific Incorporation Methodsmentioning

confidence: 99%

Unnatural amino acid mutagenesis-based enzyme engineering

Ravikumar

Nadarajan

Yoo

et al. 2015

Trends in Biotechnology

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“…For example, E. coli cells that use 21 amino acids have been used to express bacteriophages with surface displayed peptides and proteins containing nonnative amino acids [48,49]. This has been used to evolve nonnative amino acid-integrated protein binders, protein folds, and enzyme inhibitors.…”

Section: General Methodologymentioning

confidence: 99%

Biochemical analysis with the expanded genetic lexicon

2012

Anal Bioanal Chem

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The information used to build proteins is stored in the genetic material of every organism. In nature, ribosomes use 20 native amino acids to synthesize proteins in most circumstances. However, laboratory efforts to expand the genetic repertoire of living cells and organisms have successfully encoded more than 80 nonnative amino acids in E. coli, yeast, and other eukaryotic systems. The selectivity, fidelity, and site-specificity provided by the technology have enabled unprecedented flexibility in manipulating protein sequences and functions in cells. Various biophysical probes can be chemically conjugated or directly incorporated at specific residues in proteins, and corresponding analytical techniques can then be used to answer diverse biological questions. This review summarizes the methodology of genetic code expansion and its recent progress, and discusses the applications of commonly used analytical methods.

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Evolution of Proteins with Genetically Encoded “Chemical Warheads”

Cited by 72 publications

References 6 publications

High-Yield, Zero-Leakage Expression System with a Translational Switch Using Site-Specific Unnatural Amino Acid Incorporation

High-Yield, Zero-Leakage Expression System with a Translational Switch Using Site-Specific Unnatural Amino Acid Incorporation

Unnatural amino acid mutagenesis-based enzyme engineering

Biochemical analysis with the expanded genetic lexicon

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