Dataset Information

Cell-Free Protein Synthesis Using S30 Extracts from Escherichia coli RFzero Strains for Efficient Incorporation of Non-Natural Amino Acids into Proteins.

ABSTRACT: Cell-free protein synthesis is useful for synthesizing difficult targets. The site-specific incorporation of non-natural amino acids into proteins is a powerful protein engineering method. In this study, we optimized the protocol for cell extract preparation from the Escherichia coli strain RFzero-iy, which is engineered to lack release factor 1 (RF-1). The BL21(DE3)-based RFzero-iy strain exhibited quite high cell-free protein productivity, and thus we established the protocols for its cell culture and extract preparation. In the presence of 3-iodo-l-tyrosine (IY), cell-free protein synthesis using the RFzero-iy-based S30 extract translated the UAG codon to IY at various sites with a high translation efficiency of >90%. In the absence of IY, the RFzero-iy-based cell-free system did not translate UAG to any amino acid, leaving UAG unassigned. Actually, UAG was readily reassigned to various non-natural amino acids, by supplementing them with their specific aminoacyl-tRNA synthetase variants (and their specific tRNAs) into the system. The high incorporation rate of our RFzero-iy-based cell-free system enables the incorporation of a variety of non-natural amino acids into multiple sites of proteins. The present strategy to create the RFzero strain is rapid, and thus promising for RF-1 deletions of various E. coli strains genomically engineered for specific requirements.

SUBMITTER: Adachi J

PROVIDER: S-EPMC6387211 | biostudies-literature | 2019 Jan

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Cell-Free Protein Synthesis Using S30 Extracts from <i>Escherichia coli</i> RFzero Strains for Efficient Incorporation of Non-Natural Amino Acids into Proteins.

Adachi Jiro J Katsura Kazushige K Seki Eiko E Takemoto Chie C Shirouzu Mikako M Terada Takaho T Mukai Takahito T Sakamoto Kensaku K Yokoyama Shigeyuki S

International journal of molecular sciences 20190124 3

Cell-free protein synthesis is useful for synthesizing difficult targets. The site-specific incorporation of non-natural amino acids into proteins is a powerful protein engineering method. In this study, we optimized the protocol for cell extract preparation from the <i>Escherichia coli</i> strain RFzero-iy, which is engineered to lack release factor 1 (RF-1). The BL21(DE3)-based RFzero-iy strain exhibited quite high cell-free protein productivity, and thus we established the protocols for its c ...[more]

PMID: 30678326

Similar Datasets

Project description:BACKGROUND:Protein-based microarray platforms offer considerable promise as high-throughput technologies in proteomics. Particular advantages are provided by self-assembling protein microarrays and much interest centers around analysis of eukaryotic proteins and their molecular interactions. Efficient cell-free protein synthesis is paramount for the production of self-assembling protein microarrays, requiring optimal transcription, translation, and protein folding. The Escherichia coli S30 extract demonstrates high translation rates but lacks the protein-folding efficiency of its eukaryotic counterparts derived from rabbit reticulocyte and wheat germ extract. In comparison to E. coli, eukaryotic extracts, on the other hand, exhibit slower translation rates and poor overall protein yields. A cell-free expression system that synthesizes folded eukaryotic proteins in considerable yields would optimize in vitro translation for protein microarray assembly. RESULTS:Self-assembling autofluorescent protein microarrays were produced by in situ transcription and translation of chimeric proteins containing a C-terminal Green Fluorescent Protein tag. Proteins were immobilized as array elements using an anti-GFP monoclonal antibody. The amounts of correctly-folded chimeric proteins were quantified by measuring the fluorescence intensity from each array element. During cell-free expression, very little or no fluorescence was observed from GFP-tagged multidomain eukaryotic plant proteins when in vitro translation was performed with E. coli S30 extract. Improvement was seen using wheat germ extract, but fluorescence intensities were still low because of poor protein yields. A hybrid in vitro translation system, combining S30 and wheat germ extracts, produced high levels of correctly-folded proteins for most of the constructs that were tested. CONCLUSION:The results are consistent with the hypothesis that the wheat germ extract enhances the protein folding capabilities of the in vitro system by providing eukaryotic ribosomes and chaperones and, at the same time, the E. coli S30 extract, which includes an ATP regeneration system, translates the polypeptides at high rates. This hybrid cell-free expression system allows the facile production of high-yield protein arrays suitable for downstream assays.

Project description:Post-translational modifications (PTMs) of protein tyrosine (Tyr) residues can serve as a molecular fingerprint of exposure to distinct oxidative pathways and are observed in abnormally high abundance in the majority of human inflammatory pathologies. Reactive oxidants generated during inflammation include hypohalous acids and nitric oxide-derived oxidants, which oxidatively modify protein Tyr residues via halogenation and nitration, respectively, forming 3-chloroTyr, 3-bromoTyr, and 3-nitroTyr. Traditional methods for generating oxidized or halogenated proteins involve nonspecific chemical reactions that result in complex protein mixtures, making it difficult to ascribe observed functional changes to a site-specific PTM or to generate antibodies sensitive to site-specific oxidative PTMs. To overcome these challenges, we generated a system to efficiently and site-specifically incorporate chloroTyr, bromoTyr, and iodoTyr, and to a lesser extent nitroTyr, into proteins in both bacterial and eukaryotic expression systems, relying on a novel amber stop codon-suppressing mutant synthetase (haloTyrRS)/tRNA pair derived from the Methanosarcina barkeri pyrrolysine synthetase system. We used this system to study the effects of oxidation on HDL-associated protein paraoxonase 1 (PON1), an enzyme with important antiatherosclerosis and antioxidant functions. PON1 forms a ternary complex with HDL and myeloperoxidase (MPO) in vivo. MPO oxidizes PON1 at tyrosine 71 (Tyr71), resulting in a loss of PON1 enzymatic function, but the extent to which chlorination or nitration of Tyr71 contributes to this loss of activity is unclear. To better understand this biological process and to demonstrate the utility of our GCE system, we generated PON1 site-specifically modified at Tyr71 with chloroTyr and nitroTyr in Escherichia coli and mammalian cells. We demonstrate that either chlorination or nitration of Tyr71 significantly reduces PON1 enzymatic activity. This tool for site-specific incorporation of halotyrosine will be critical to understanding how exposure of proteins to hypohalous acids at sites of inflammation alters protein function and cellular physiology. In addition, it will serve as a powerful tool for generating antibodies that can recognize site-specific oxidative PTMs.

Dataset Information

Cell-Free Protein Synthesis Using S30 Extracts from Escherichia coli RFzero Strains for Efficient Incorporation of Non-Natural Amino Acids into Proteins.

Publications

Cell-Free Protein Synthesis Using S30 Extracts from <i>Escherichia coli</i> RFzero Strains for Efficient Incorporation of Non-Natural Amino Acids into Proteins.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets