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Cloning, molecular characterization and expression of a cDNA encoding a functional NADH-cytochrome b5 reductase from Mucor racemosus PTCC 5305 in E. coli
Setayesh, Ned A; Sepehrizadeh, Zargham; Jaberi, Elham; Yazdi, Mojtaba Tabatabaei.
  • Setayesh, Ned A; Tehran University of Medical Sciences. Faculty of Pharmacy. Department of Pharmaceutical Biotechnology. Laboratory of Genetic Engineering. Tehran. IR
  • Sepehrizadeh, Zargham; Tehran University of Medical Sciences. Faculty of Pharmacy. Department of Pharmaceutical Biotechnology. Laboratory of Genetic Engineering. Tehran. IR
  • Jaberi, Elham; Tehran University of Medical Sciences. Faculty of Pharmacy. Department of Pharmaceutical Biotechnology. Laboratory of Genetic Engineering. Tehran. IR
  • Yazdi, Mojtaba Tabatabaei; Tehran University of Medical Sciences. Faculty of Pharmacy. Department of Pharmaceutical Biotechnology. Laboratory of Genetic Engineering. Tehran. IR
Biol. Res ; 42(2): 137-146, 2009. ilus, tab
Article in English | LILACS | ID: lil-524883
ABSTRACT
The present work aims to study a new NADH-cytochrome b5 reductase (cb5r) from Mucor racemosus PTCC 5305. A cDNA coding for cb s r was isolated from a Mucor racemosus PTCC 5305 cDNA library. The nucleotide sequence of the cDNA including coding and sequences flanking regions was determined. The open reading frame starting from ATG and ending with TAG stop codon encoded 228 amino acids and displayed the closest similarity (73 percent) with Mortierella alpina cb s r. Lack of hydrophobic residues in the N-terminal sequence was apparent, suggesting that the enzyme is a soluble isoform. The coding sequence was then cloned in the pET16b transcription vector carrying an N-terminal-linked His-Tag® sequence and expressed in Escherichia coli BL21 (DE3). The enzyme was then homogeneously purified by a metal affinity column. The recombinant Mucor enzyme was shown to have its optimal activity at pH and temperature of about 7.5 and 40 °C, respectively. The apparent Km value was calculated to be 13 μM for ferricyanide. To our knowledge, this is the first report on cloning and expression of a native fungal soluble isoform of NADH-cytochrome b5 reductase in E. coli.
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Full text: Available Index: LILACS (Americas) Main subject: DNA, Complementary / Escherichia coli / Genetic Vectors / Mucor Language: English Journal: Biol. Res Journal subject: Biology Year: 2009 Type: Article / Project document Affiliation country: Iran Institution/Affiliation country: Tehran University of Medical Sciences/IR

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Full text: Available Index: LILACS (Americas) Main subject: DNA, Complementary / Escherichia coli / Genetic Vectors / Mucor Language: English Journal: Biol. Res Journal subject: Biology Year: 2009 Type: Article / Project document Affiliation country: Iran Institution/Affiliation country: Tehran University of Medical Sciences/IR