Characterization of homogeneous recombinant glutaredoxin from Escherichia coli: purification from an inducible lambda PL expression system and properties of a novel elongated form.
We have constructed a plasmid, pAHOB1, with a 482-b AluI fragment containing the Escherichia coli glutaredoxin gene (grx) cloned under lambda PL promoter control. Growth of E. coli N4830/pAHOB1 cells at 30 degrees C followed by heat induction at 40 degrees C for 5 h resulted in expression of glutaredoxin as 20% of the soluble E. coli protein. Methods for the preparation of gram amounts of glutaredoxin and 5 mM solutions suitable for NMR studies were developed. About 10% of the glutaredoxin activity showed an unexpected higher isoelectric point and was isolated by DEAE-cellulose chromatography at pH 6.0. Sequence analysis demonstrated that this novel form (grx-90) contained five additional N-terminal residues (Met-Arg-Arg-Glu-Ile) added to the glutaredoxin molecule with 85 residues (grx-85). Grx-90 originates from an alternative ATG initiation codon present 5' of the previously identified translation start site on the grx gene in E. coli. Despite the highly charged N-terminal extension, grx-90 showed full activity as a GSH-disulfide oxidoreductase and the same apparent Km value (0.14 microM) as glutaredoxin in GSH-dependent reduction of CDP by ribonucleotide reductase. Both grx-90 and grx-85 showed identical competition curves in radioimmunoassays. The presence of grx-90 was also demonstrated in log-phase E. coli C600 cells as 5 to 10% of total glutaredoxin by immunological techniques. The molar extinction coefficient of native glutaredoxin (12,500 M-1 cm-1 at 280 nm) was 15% higher than expected from its content of one Trp and four Tyr residues.
Björnberg
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O
Department of Physiological Chemistry, Karolinska Institutet, Stockholm, Sweden.
Holmgren
A
A
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D005798:Genes, Bacterial; D054477:Glutaredoxins; D007525:Isoelectric Focusing; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010088:Oxidoreductases; D010957:Plasmids; D011506:Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D012995:Solubility
D004269:DNA, Bacterial; D054477:Glutaredoxins; D011506:Proteins; D011994:Recombinant Proteins; D010088:Oxidoreductases