An improved system for the generation and analysis of mutant proteins containing unnatural amino acids in Saccharomyces cerevisiae.

An improved system for the generation and analysis of mutant proteins containing unnatural amino acids in Saccharomyces cerevisiae. Research Abstract Details 

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An improved system for the generation and analysis of mutant proteins containing unnatural amino acids in Saccharomyces cerevisiae. Abstract Text:

Shawn Chen,Peter G Schultz,Ansgar Brock,

We have previously described methodology that makes it possible to genetically encode a wide array of unnatural amino acids in both prokaryotic and eukaryotic organisms. Here, we report the systematic optimization of a Saccharomyces cerevisiae expression system for the production of mutant proteins containing unnatural amino acids. Modifications include significant increases in both the expression levels of the orthogonal Escherichia coli amber suppressor tRNA(CUA) and cognate aminoacyl-tRNA synthetase (aaRS) pair, and expression of the target protein gene using a strong transcriptional promoter, optimized codons and elevated plasmid copy numbers. With this new system, a number of unnatural amino acids, including the photocrosslinkers p-benzoylphenylalanine and p-azidophenylalanine, and the chemically reactive amino acids, p-acetylphenylalanine and p-propargyloxyphenylalanine, were incorporated into human superoxide dismutase (hSOD) in yeast in good yields (maximally approximately 6-8 mg/l of culture in most cases). Mass spectrometric analysis of the hSOD mutants was performed with high dynamic range using multiple reaction monitoring that provided new insights into the factors that control the fidelity of unnatural amino acid incorporation.

An improved system for the generation and analysis of mutant proteins containing unnatural amino acids in Saccharomyces cerevisiae. Publishing Authors By Initials

S Chen,PG Schultz,A Brock,

For similar enzymes and coenzymes: enzymes: oxidoreductases: superoxide dismutase research abstracts see: enzymes and coenzymes: enzymes: oxidoreductases: superoxide dismutase research

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An improved system for the generation and analysis of mutant proteins containing unnatural amino acids in Saccharomyces cerevisiae. Journal Published:

PUBLICATION TYPE: Research Support, Non-U.S. Gov

Journal: Journal of molecular biology

VOLUME: 371

Page Numbers: 112-22

Journal Abbreviation: J. Mol. Biol.

ISSN: 0022-2836

DAY: 22

MONTH: 05

YEAR: 2007

An improved system for the generation and analysis of mutant proteins containing unnatural amino acids in Saccharomyces cerevisiae. Information

Number of References:

LANGUAGE: eng

NlmUniqueID: 2985088

An improved system for the generation and analysis of mutant proteins containing unnatural amino acids in Saccharomyces cerevisiae. Keywords Mesh Terms:

KEYWORDS: Superoxide Dismutase

MESH TERMS: metabolism

Chemical & Substance for Abstract: An improved system for the generation and analysis of mutant proteins containing unnatural amino acids in Saccharomyces cerevisiae. Information

Substance Name: Amino Acyl-tRNA Synthetases

Registry Number: EC 6.1.1.-

Grant and Affiliation Information for An improved system for the generation and analysis of mutant proteins containing unnatural amino acids in Saccharomyces cerevisiae.

AFFILIATION: Department of Chemistry and the Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.

Country: England

AGENCY: United States NIGMS

GRANT: GM62159

ACRONYM: GM

MEDLINETA: J Mol Biol

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