Cloning, expression and purification of Phospholipase from Natrialba asiatica

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.

کلیدواژه ها:

phospholipase ، Cloning ، Expression ، Natrialba asiatica ، pET-28a