Large scale production of biologically active Escherichia coli glutamyl-tRNA reductase from inclusion bodies.
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.
Schauer
Stefan
S
Institute of Microbiology, Technical University Braunschweig, Spielmannstrasse 7, D-38106 Braunschweig, Germany.
Lüer
Corinna
C
Moser
Jürgen
J
D000445:Aldehyde Oxidoreductases / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D000818:Animals; D002850:Chromatography, Gel; D002942:Circular Dichroism; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D002479:Inclusion Bodies / Q000378:metabolism; D010957:Plasmids; D011487:Protein Conformation; D015202:Protein Engineering; D017510:Protein Folding; D015854:Up-Regulation
D000445:Aldehyde Oxidoreductases; C067282:glutamyl tRNA reductase