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Development of high-yield autofluorescent protein microarrays using hybrid cell-free expression with combined Escherichia coli S30 and wheat germ extracts.

ABSTRACT: 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.

SUBMITTER: Zarate X

PROVIDER: S-EPMC2906421 | biostudies-literature | 2010 Jun

REPOSITORIES: biostudies-literature

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Development of high-yield autofluorescent protein microarrays using hybrid cell-free expression with combined Escherichia coli S30 and wheat germ extracts.

Zárate Xristo X Henderson David C DC Phillips Keenan C KC Lake April D AD Galbraith David W DW

Proteome science 20100615

<h4>Background</h4>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 S ...[more]

PMID: 20546627

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Project description:Understanding the regional relationships between climate change and crop production will benefit strategic decisions for future agricultural adaptation in China. In this study, the combined effects of climatic factors on spring wheat phenophase and grain yield over the past three decades in Inner Mongolia, China, were explored based on the daily climate variables from 1981-2014 and detailed observed data of spring wheat from 1981-2014. Inner Mongolia was divided into three different climate type regions, the eastern, central and western regions. The data were gathered from 10 representative agricultural meteorological experimental stations in Inner Mongolia and analysed with the Agricultural Production Systems Simulator (APSIM) model. First, the performance of the APSIM model in the spring wheat planting areas of Inner Mongolia was tested. Then, the key climatic factors limiting the phenophases and yield of spring wheat were identified. Finally, the responses of spring wheat phenophases and yield to climate change were further explored regionally. Our results revealed a general yield reduction of spring wheat in response to the pronounced climate warming from 1981 to 2014, with an average of 3564 kg·ha-1. The regional differences in yields were significant. The maximum potential yield of spring wheat was found in the western region. However, the minimum potential yield was found in the middle region. The air temperature and soil surface temperature were the optimum climatic factors that affected the key phenophases of spring wheat in Inner Mongolia. The influence of the average maximum temperature on the key phenophases of spring wheat was greater than the average minimum temperature, followed by the relative humidity and solar radiation. The most insensitive climatic factors were precipitation, wind speed and reference crop evapotranspiration. As for the yield of spring wheat, temperature, solar radiation and air relative humidity were major meteorological factors that affected in the eastern and western Inner Mongolia. Furthermore, the effect of the average minimum temperature on yield was greater than that of the average maximum temperature. The increase of temperature in the western and middle regions would reduce the spring wheat yield, while in the eastern region due to the rising temperature, the spring wheat yield increased. The increase of solar radiation in the eastern and central regions would increase the yield of spring wheat. The increased air relative humidity would make the western spring wheat yield increased and the eastern spring wheat yield decreased. Finally, the models describing combined effects of these dominant climatic factors on the maturity and yield in different regions of Inner Mongolia were used to establish geographical differences. Our findings have important implications for improving climate change impact studies and for local agricultural production to cope with ongoing climate change.

Dataset Information

Development of high-yield autofluorescent protein microarrays using hybrid cell-free expression with combined Escherichia coli S30 and wheat germ extracts.

Publications

Development of high-yield autofluorescent protein microarrays using hybrid cell-free expression with combined Escherichia coli S30 and wheat germ extracts.

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