Abstract
We have improved the productivity of an Escherichia coli cell-free protein synthesis system. First, creatine phosphate and creatine kinase were used as the energy source regeneration system, and the other components of the reaction mixture were optimized. Second, the E. coli S30 cell extract was condensed by dialysis against a polyethylene glycol solution to increase the rate of synthesis. Third, during the protein synthesis, the reaction mixture was dialyzed against a low-molecular-weight substrate solution to prolong the reaction. Thus, the yield of chloramphenicol acetyltransferase was raised to 6 mg/ml of reaction mixture. Stable-isotope labeling of a protein with 13C/15N-labeled amino acids for NMR spectroscopy was achieved by this method.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Biotechnology
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Carbon Isotopes
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Chloramphenicol O-Acetyltransferase / biosynthesis
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Chloramphenicol O-Acetyltransferase / chemistry
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Chloramphenicol O-Acetyltransferase / genetics
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Creatine Kinase / metabolism
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Dialysis
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Escherichia coli / genetics
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Escherichia coli / metabolism
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Humans
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Kinetics
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Magnetic Resonance Spectroscopy
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Nitrogen Isotopes
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Phosphocreatine / metabolism
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Recombinant Proteins / biosynthesis*
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Recombinant Proteins / chemistry
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Recombinant Proteins / genetics
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ras Proteins / biosynthesis
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ras Proteins / chemistry
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ras Proteins / genetics
Substances
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Carbon Isotopes
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Nitrogen Isotopes
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Recombinant Proteins
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Phosphocreatine
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Chloramphenicol O-Acetyltransferase
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Creatine Kinase
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ras Proteins