COI code: CIGS15_608
Paper Language: English
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Authors Cloning, expression and purification of Phospholipase from Natrialba asiaticaFatemeh Allami Mehmandoosti - Department of Biology, Islamic Azad University, Science and Research Branch, Tehran, Iran
Amir Amiri - Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran
Farangis Ataei - Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran
Mehdi Ebrahimi - Department of Biochemistry & amp; Biophysics, School of Biological Sciences, Islamic Azad University, Varamin-Pishva Branch,Tehran, Iran
Abstract:Phospholipases (EC 3.1.1 Carboxylesterase) are interfacial enzymes that hydrolyze hydrophobic ester linkages of triacylglycerols and phospholipids. In addition to their role as esterases, these enzymes catalyze other reactions such as esterification, transesterification and interesterification. Microbial phospholipases are preferred to those derived from animals and plants. Phospholipases are used in various industrial, such as for biodiesels, food, nutraceuticals, oil degumming and detergents, also include bioremediation, agriculture, cosmetics, leather and paper industries. In the present investigation, phospholipase from halophilic archaea Natrialba asiatica was amplified by PCR using specific primers (containing restriction sites EcoRI and HindIII). Then pET-28a as cloning vector was extracted. Next pET-28a and phospholipase gene were digested by restriction enzymes and ligated. PET-28a containing phospholipase was transformed into E.coli DH5α cells. Screening carried out using LB-agar plates containing kanamycin. After double digestion, the nucleotide sequencing was confirmed using universal T7 promoter by macrogene Korea. The confirmed gene was transferred into E.coli BL21, and cultured up to OD600-nm ~2/5 in LB medium. Then was added 0.5mM IPTG and induced for 45 min at 37 0C. Recombinant His-tagged protein purified by affinity chromatography demonstrated a band about 35 kDa on 12.5 % SDS-PAGE gel.
Keywords:phospholipase, Cloning, Expression, Natrialba asiatica, pET-28a
COI code: CIGS15_608
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Allami Mehmandoosti, Fatemeh; Amir Amiri; Farangis Ataei & Mehdi Ebrahimi, 2018, Cloning, expression and purification of Phospholipase from Natrialba asiatica, The Third International and 15th National Genetics Congress, تهران, انجمن علمي ژنتيك ايران, https://www.civilica.com/Paper-CIGS15-CIGS15_608.htmlInside the text, wherever referred to or an achievement of this article is mentioned, after mentioning the article, inside the parental, the following specifications are written.
First Time: (Allami Mehmandoosti, Fatemeh; Amir Amiri; Farangis Ataei & Mehdi Ebrahimi, 2018)
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The University/Research Center Information:
Type: Azad University
Paper No.: 22199
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