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Heterologous Expression, Purification and Characterization of a Peroxidase Isolated from Lepidium draba

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Abstract

Peroxidase is one of the most widely used enzymes in biotechnology and medicine. In the current study, cDNA encoding peroxidase from Lepidium draba (LDP) was cloned and expressed in Escherichia coli BL21 (DE3) cells in the form of inclusion bodies (IBs). To achieve purified active enzyme, IBs were solubilized before being purified and refolded. The deduced amino acid sequence (308) of the LDP gene (924 bp) revealed 88.96% identity to horseradish peroxidase C1A (HRP C1A). The results of basic local alignment search tool (BLAST) and phylogenetic analysis of the protein sequence showed that this enzyme belongs to the neutral group of class III plant peroxidases. According to sequence analysis and structural modeling, critical amino acids in heme and calcium binding domain as well as cysteine residues were conserved as HRP C1A except for calcium binding domain where valine228 was replaced with isoleucine. The far-UV circular dichroism (CD) results were confirmed by homology modeling data showing the enzyme consists mainly of α-helices as other plant peroxidases. Overall, according to the results of catalytic activity and refolding yield, LDP can be introduced as a novel peroxidase for medical and biotechnology applications.

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Abbreviations

CD:

Circular dichroism

DTT:

Dithiotheritol

HRP:

Horseradish peroxidase

IBs:

Inclusion bodies

LDP:

Lepidium draba peroxidase

K m :

Michaelis–Menten constant

PCR:

Polymerase chain reaction

RZ :

Reinheitzahl

SDS-PAGE:

Sodium dodecyl sulfate polyacrylamide gel electrophoresis

TMB:

3,3′,5,5′-tetramethylbenzidine

V max :

Maximum velocity

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Acknowledgements

Financial support for this work was provided by Research Council of Jiroft University of Medical Sciences under contract number of p-94-10.

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Authors and Affiliations

  1. Department of Biotechnology, Institute of Science and High Technology and Environmental Sciences, Graduate University of Advanced Technology, Highway Haft Bagh Alavi, PO Box 76315–117, Kerman, Iran

    Yaser Fattahian, Ali Riahi-Madvar & Masoud Torkzadeh-Mahani

  2. Jiroft University of Medical Sciences, Jiroft, Iran

    Reza Mirzaee

  3. Endocrinology and Metabolism Research Center, Institute of Basic and Clinical Physiology Sciences, Kerman University of Medical Sciences, Kerman, Iran

    Gholamreza Asadikaram

  4. Department of Biochemistry, School of Medicine, Kerman University of Medical Sciences, Kerman, Iran

    Gholamreza Asadikaram

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  1. Yaser Fattahian
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  2. Ali Riahi-Madvar
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Correspondence to Ali Riahi-Madvar.

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Fattahian, Y., Riahi-Madvar, A., Mirzaee, R. et al. Heterologous Expression, Purification and Characterization of a Peroxidase Isolated from Lepidium draba . Protein J 36, 461–471 (2017). https://doi.org/10.1007/s10930-017-9741-y

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