Abstract: The present disclosure describes methods and systems for improving the expression of a properly folded, biologically active protein of interest in a cell free synthesis system. The methods and systems use a bacterial cell free extract having an active oxidative phosphorylation system, and include an exogenous protein chaperone. The exogenous protein chaperone can be expressed by the bacteria used to prepare the cell free extract. The exogenous protein chaperone can be a protein disulfide isomerase and/or a peptidyl-prolyl cis-trans isomerase. The inventors discovered that the combination of a protein disulfide isomerase and a peptidyl-prolyl cis-trans isomerase produces a synergistic increase in the amount of properly folded, biologically active protein of interest.

Abstract: The present disclosure describes methods and systems for improving the expression of a properly folded, biologically active protein of interest in a cell free synthesis system. The methods and systems use a bacterial cell free extract having an active oxidative phosphorylation system, and include an exogenous protein chaperone. The exogenous protein chaperone can be expressed by the bacteria used to prepare the cell free extract. The exogenous protein chaperone can be a protein disulfide isomerase and/or a peptidyl-prolyl cis-trans isomerase. The inventors discovered that the combination of a protein disulfide isomerase and a peptidyl-prolyl cis-trans isomerase produces a synergistic increase in the amount of properly folded, biologically active protein of interest.

Abstract: This invention provides high growth capacity strains of auxotrophic Escherichia coli and methods for generating thereof. The high growth capacity strains express a complementing auxotrophic plasmid that allows the strain to grow in the absence of the auxotrophic amino acid. Also, provided herein is a method for preparing a bacterial cell extract of a high growth capacity strain of auxotrophic Escherichia coli for use in an in vitro protein expression.

Abstract: This invention provides high growth capacity strains of auxotrophic Escherichia coli and methods for generating thereof. The high growth capacity strains express a complementing auxotrophic plasmid that allows the strain to grow in the absence of the auxotrophic amino acid. Also, provided herein is a method for preparing a bacterial cell extract of a high growth capacity strain of auxotrophic Escherichia coli for use in an in vitro protein expression.

Abstract: The present disclosure describes methods and systems for improving the expression of a properly folded, biologically active protein of interest in a cell free synthesis system. The methods and systems use a bacterial cell free extract having an active oxidative phosphorylation system, and include an exogenous protein chaperone. The exogenous protein chaperone can be expressed by the bacteria used to prepare the cell free extract. The exogenous protein chaperone can be a protein disulfide isomerase and/or a peptidyl-prolyl cis-trans isomerase. The inventors discovered that the combination of a protein disulfide isomerase and a peptidyl-prolyl cis-trans isomerase produces a synergistic increase in the amount of properly folded, biologically active protein of interest.

Abstract: The present disclosure describes methods and systems for improving the expression of a properly folded, biologically active protein of interest in a cell free synthesis system. The methods and systems use a bacterial cell free extract having an active oxidative phosphorylation system, and include an exogenous protein chaperone. The exogenous protein chaperone can be expressed by the bacteria used to prepare the cell free extract. The exogenous protein chaperone can be a protein disulfide isomerase and/or a peptidyl-prolyl cis-trans isomerase. The inventors discovered that the combination of a protein disulfide isomerase and a peptidyl-prolyl cis-trans isomerase produces a synergistic increase in the amount of properly folded, biologically active protein of interest.

Abstract: This invention provides high growth capacity strains of auxotrophic Escherichia coli and methods for generating thereof. The high growth capacity strains express a complementing auxotrophic plasmid that allows the strain to grow in the absence of the auxotrophic amino acid. Also, provided herein is a method for preparing a bacterial cell extract of a high growth capacity strain of auxotrophic Escherichia coli for use in an in vitro protein expression.

Abstract: The invention relates to orthogonal pairs of tRNAs and aminoacyl-tRNA synthetase that can incorporate unnatural amino acid into proteins produced in eubacterial host cells such as E. coli, or in a eukaryotic host such as a yeast cell. The invention provides, for example but not limited to, novel orthogonal synthetases, methods for identifying and making the novel synthetases, methods for producing proteins containing unnatural amino acids, and translation systems.

Abstract: The present disclosure describes methods and systems for improving the expression of a properly folded, biologically active protein of interest in a cell free synthesis system. The methods and systems use a bacterial cell free extract having an active oxidative phosphorylation system, and include an exogenous protein chaperone. The exogenous protein chaperone can be expressed by the bacteria used to prepare the cell free extract. The exogenous protein chaperone can be a protein disulfide isomerase and/or a peptidyl-prolyl cis-trans isomerase. The inventors discovered that the combination of a protein disulfide isomerase and a peptidyl-prolyl cis-trans isomerase produces a synergistic increase in the amount of properly folded, biologically active protein of interest.

Abstract: The invention relates to orthogonal pairs of tRNAs and aminoacyl-tRNA synthetase that can incorporate unnatural amino acid into proteins produced in eubacterial host cells such as E. coli, or in a eukaryotic host such as a yeast cell. The invention provides, for example but not limited to, novel orthogonal synthetases, methods for identifying and making the novel synthetases, methods for producing proteins containing unnatural amino acids, and translation systems.

Abstract: Compositions and methods of producing components of protein biosynthetic machinery that include orthogonal leucyl-tRNAs, orthogonal leucyl-aminoacyl-tRNA synthetases, and orthogonal pairs of leucyl-tRNAs/synthetases, which incorporate photoregulated amino acids, OMe-L-tyrosine, ?-aminocaprylic acid, or o-nitrobenzyl cysteine into proteins are proteins are provided in response to an amber selector codon. Methods for identifying these orthogonal pairs are also provided along with methods of producing proteins with a photoregulated amino acid, Ome-L-tyrosine, ?-aminocaprylic acid, or o-nitrobenzyl cysteine using these orthogonal pairs.

Abstract: The invention relates to orthogonal pairs of tRNAs and aminoacyl-tRNA synthetases that can incorporate unnatural amino acids into proteins produced in eubacterial host cells such as E. coli, or in a eukaryotic host such as a yeast cell. The invention provides, for example but not limited to, novel orthogonal synthetases, methods for identifying and making the novel synthetases, methods for producing proteins containing unnatural amino acids, and translation systems.

Abstract: Compositions and methods of producing components of protein biosynthetic machinery that include orthogonal leucyl-tRNAs, orthogonal leucyl-aminoacyl-tRNA synthetases, and orthogonal pairs of leucyl-tRNAs/synthetases, which incorporate photoregulated amino acids, OMe-L-tyrosine, ?-aminocaprylic acid, or o-nitrobenzyl cysteine into proteins are provided in response to an amber selector codon. Methods for identifying these orthogonal pairs are also provided along with methods of producing proteins with a photoregulated amino acid, OMe-L-tyrosine, ?-aminocaprylic acid, or o-nitrobenzyl cysteine using these orthogonal pairs.