title: Cloning, molecular characterization and expression of a cDNA encoding a functional NADH-cytochrome b5 reductase from Mucor racemosus PTCC 5305 in E. coli.
abstract: The present work aims to study a new NADH-cytochrome b5 reductase (cb5r) from Mucor racemosus PTCC 5305. A cDNA coding for cb5r 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%) with Mortierella alpina cb5r. 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 degrees C, respectively. The apparent K(m) value was calculated to be 13 microM 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.
authors.affiliation: Laboratory of Genetic Engineering, Department of Pharmaceutical Biotechnology, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran 14174, Iran.
authors.forename: Neda
authors.initials: N
authors.lastname: Setayesh
authors.forename: Zargham
authors.initials: Z
authors.lastname: Sepehrizadeh
authors.forename: Elham
authors.initials: E
authors.lastname: Jaberi
authors.forename: Mojtaba Tabatabaei
authors.initials: MT
authors.lastname: Yazdi
mesh_terms: D001483:Base Sequence; D003001:Cloning, Molecular; D042966:Cytochrome-B(5) Reductase / Q000235:genetics* / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics*; D015723:Gene Library; D005822:Genetic Vectors / Q000235:genetics*; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009089:Mucor / Q000201:enzymology*; D016366:Open Reading Frames; D014158:Transcription, Genetic
chemical_list: D018076:DNA, Complementary; D007527:Isoenzymes; D042966:Cytochrome-B(5) Reductase