Cloning, molecular characterization and expression of a cDNA encoding a functional NADH-cytochrome b5 reductase from Mucor racemosus PTCC 5305 in E. coli

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 r...

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Autores principales:	SETAYESH,NED A, SEPEHRIZADEH,ZARGHAM, JABERI,ELHAM, YAZDI,MOJTABA TABATABAEI
Lenguaje:	English
Publicado:	Sociedad de Biología de Chile 2009
Materias:	Mucor racemosus NADH-cytochrome b s reductase soluble isoform
Acceso en línea:	http://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0716-97602009000200001
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spelling	oai:scielo:S0716-976020090002000012009-10-09Cloning, molecular characterization and expression of a cDNA encoding a functional NADH-cytochrome b5 reductase from Mucor racemosus PTCC 5305 in E. coliSETAYESH,NED ASEPEHRIZADEH,ZARGHAMJABERI,ELHAMYAZDI,MOJTABA TABATABAEI Mucor racemosus NADH-cytochrome b s reductase soluble isoform 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%) 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.info:eu-repo/semantics/openAccessSociedad de Biología de ChileBiological Research v.42 n.2 20092009-01-01text/htmlhttp://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0716-97602009000200001en10.4067/S0716-97602009000200001
institution	Scielo Chile
collection	Scielo Chile
language	English
topic	Mucor racemosus NADH-cytochrome b s reductase soluble isoform
spellingShingle	Mucor racemosus NADH-cytochrome b s reductase soluble isoform SETAYESH,NED A SEPEHRIZADEH,ZARGHAM JABERI,ELHAM YAZDI,MOJTABA TABATABAEI Cloning, molecular characterization and expression of a cDNA encoding a functional NADH-cytochrome b5 reductase from Mucor racemosus PTCC 5305 in E. coli
description	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%) 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.
author	SETAYESH,NED A SEPEHRIZADEH,ZARGHAM JABERI,ELHAM YAZDI,MOJTABA TABATABAEI
author_facet	SETAYESH,NED A SEPEHRIZADEH,ZARGHAM JABERI,ELHAM YAZDI,MOJTABA TABATABAEI
author_sort	SETAYESH,NED A
title	Cloning, molecular characterization and expression of a cDNA encoding a functional NADH-cytochrome b5 reductase from Mucor racemosus PTCC 5305 in E. coli
title_short	Cloning, molecular characterization and expression of a cDNA encoding a functional NADH-cytochrome b5 reductase from Mucor racemosus PTCC 5305 in E. coli
title_full	Cloning, molecular characterization and expression of a cDNA encoding a functional NADH-cytochrome b5 reductase from Mucor racemosus PTCC 5305 in E. coli
title_fullStr	Cloning, molecular characterization and expression of a cDNA encoding a functional NADH-cytochrome b5 reductase from Mucor racemosus PTCC 5305 in E. coli
title_full_unstemmed	Cloning, molecular characterization and expression of a cDNA encoding a functional NADH-cytochrome b5 reductase from Mucor racemosus PTCC 5305 in E. coli
title_sort	cloning, molecular characterization and expression of a cdna encoding a functional nadh-cytochrome b5 reductase from mucor racemosus ptcc 5305 in e. coli
publisher	Sociedad de Biología de Chile
publishDate	2009
url	http://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0716-97602009000200001
work_keys_str_mv	AT setayeshneda cloningmolecularcharacterizationandexpressionofacdnaencodingafunctionalnadhcytochromeb5reductasefrommucorracemosusptcc5305inecoli AT sepehrizadehzargham cloningmolecularcharacterizationandexpressionofacdnaencodingafunctionalnadhcytochromeb5reductasefrommucorracemosusptcc5305inecoli AT jaberielham cloningmolecularcharacterizationandexpressionofacdnaencodingafunctionalnadhcytochromeb5reductasefrommucorracemosusptcc5305inecoli AT yazdimojtabatabatabaei cloningmolecularcharacterizationandexpressionofacdnaencodingafunctionalnadhcytochromeb5reductasefrommucorracemosusptcc5305inecoli
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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

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