PMID:14550647
| Citation |
Schauer, S, Lüer, C and Moser, J (2003) Large scale production of biologically active Escherichia coli glutamyl-tRNA reductase from inclusion bodies. Protein Expr. Purif. 31:271-5 |
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| Abstract |
Glutamyl-tRNA reductase catalyzes the initial step of tetrapyrrole biosynthesis in plants and prokaryotes. Recombinant Escherichia coli glutamyl-tRNA reductase was purified to apparent homogeneity from an overproducing E. coli strain by a two-step procedure yielding 5.6 mg of enzyme per gram of wet cells with a specific activity of 0.47 micromol min(-1)mg(-1). After recombinant production, denatured glutamyl-tRNA reductase from inclusion bodies was renatured by an on-column refolding procedure. Residual protein aggregates were removed using Superdex 200 gel-filtration chromatography. Solubility, specific activity, and long-term storage properties were improved compared to previous protocols. Obtained enzyme amounts of high purity now allow the research on the recognition mechanism of tRNAGlu and high-throughput inhibitor screening. |
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| Keywords |
Aldehyde Oxidoreductases/biosynthesis; Aldehyde Oxidoreductases/genetics; Aldehyde Oxidoreductases/metabolism; Animals; Chromatography, Gel; Circular Dichroism; Escherichia coli/enzymology; Escherichia coli/genetics; Escherichia coli/metabolism; Gene Expression Regulation; Genetic Vectors; Inclusion Bodies/metabolism; Plasmids; Protein Conformation; Protein Engineering; Protein Folding; Up-Regulation |
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