High-yield cell-free protein synthesis for site-specific incorporation of unnatural amino acids at two sites

Research School of Chemistry, Australian National University, Canberra, ACT 0200, Australia.
Biochemical and Biophysical Research Communications (Impact Factor: 2.28). 02/2012; 418(4):652-6. DOI: 10.1016/j.bbrc.2012.01.069
Source: PubMed

ABSTRACT Using aminoacyl-tRNA synthetase/suppressor tRNA pairs derived from Methanocaldococcus jannaschii, an Escherichia coli cell-free protein production system affords proteins with site-specifically incorporated unnatural amino acids (UAAs) in high yields through the use of optimized amber suppressor tRNA(CUA)(opt) and optimization of reagent concentrations. The efficiency of the cell-free system allows the incorporation of trifluoromethyl-phenylalanine using a polyspecific synthetase evolved previously for p-cyano-phenylalanine, and the incorporation of UAAs at two different sites of the same protein without any re-engineering of the E. coli cells used to make the cell-free extract.

  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Residue specific incorporation of non-canonical amino acids into proteins is usually performed in vivo using amino acid auxotrophic strains and replacing the natural amino acid with an unnatural amino acid analog. Herein, we present an efficient amino acid depleted cell-free protein synthesis system that can be used to study residue specific replacement of a natural amino acid by an unnatural amino acid analog. This system combines a simple methodology and high protein expression titers with a high efficiency analog substitution into a target protein. To demonstrate the productivity and efficacy of a cell-free synthesis system for residue-specific incorporation of unnatural amino acids in vitro, we use this system to show that 5-fluorotryptophan and 6-fluorotryptophan substituted streptavidin retain the ability to bind biotin despite protein-wide replacement of a natural amino acid for the amino acid analog. We envisage this amino acid-depleted cell-free synthesis system being an economical and convenient format for the high-throughput screening of a myriad of amino acid analogs with a variety of protein targets for the study and functional characterization of proteins substituted with unnatural amino acids when compared to the currently employed in vivo methodologies.
    Journal of Biotechnology 05/2014; 178. DOI:10.1016/j.jbiotec.2014.02.009 · 2.88 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Site-specific incorporation of non-standard amino acids (NSAAs) into proteins opens the way to novel biological insights and applications in biotechnology. Here, we describe the development of a high yielding cell-free protein synthesis (CFPS) platform for NSAA incorporation from crude extracts of genomically recoded Escherichia coli lacking release factor 1. We used genome engineering to construct synthetic organisms that, upon cell lysis, lead to improved extract performance. We targeted five potential negative effectors to be disabled: the nuclease genes rna, rnb, csdA, mazF, and endA. Using our most productive extract from strain MCJ.559 (csdA(-) endA(-) ), we synthesized 550±40 μg mL(-1) of modified superfolder green fluorescent protein containing p-acetyl-L-phenylalanine. This yield was increased to ∼1300 μg mL(-1) when using a semicontinuous method. Our work has implications for using whole genome editing for CFPS strain development, expanding the chemistry of biological systems, and cell-free synthetic biology. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
    ChemBioChem 03/2015; 16(5). DOI:10.1002/cbic.201402708 · 3.06 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Antibody conjugates are important in many areas of medicine and biological research, and antibody drug conjugates (ADCs) are becoming an important next generation class of therapeutics for cancer treatment. Early conjugation technologies relied upon random conjugation to multiple amino acid side chains, resulting in heterogenous mixtures of labeled antibody. Recent studies, however, strongly support the notion that site-specific conjugation produces a homogenous population of antibody conjugates with improved pharmacologic properties over randomly coupled molecules. Genetically incorporated unnatural amino acids (uAAs) allow unique orthogonal coupling strategies compared to thosed used for the naturally occuring twenty amino acids. Thus uAAs provide a novel paradigm for creation of next generation ADCs. Additionally, uAA-based site-specific conjugation could also empower creation of additional multifunctional conjugates important as biopharmaceuticals, diagnostics, or reagents.
    Molecular Pharmaceutics 04/2015; DOI:10.1021/acs.molpharmaceut.5b00082 · 4.79 Impact Factor