Cloning and Cytoplasmic Expression of L-Asparaginase II in E. coli
Subject Areas : Microbial BiotechnologyHossein Mahboudi 1 , Mansour Abachi 2 , Shahram Araghi 3 , Haleh Hamedifar 4 , Behrouz Vaziri 5 , Fereidoun Mahboudi 6
1 - Department of Microbiology, Qom Branch, Islamic Azad University, Qom, Iran
2 - Department of Research and Development, CinnaGen Co., Tehran, Iran
3 - Department of Research and Development, CinnaGen Co., Tehran, Iran
4 - Department of Research and Development, CinnaGen Co., Tehran, Iran
5 - Biotechnology Research Center, Protein Chemistry Unit, Pasteur Institute of Iran, Tehran, Iran
6 - Biotechnology Research Center, Recombinant Protein Unit, Pasteur Institute of Iran, Tehran, Iran
Keywords: Escherichia coli, Cloning, Gene expression, L-Asparaginase II,
Abstract :
Background and objectives: L-Asparaginase (isozyme II) is a natural product of E. coli that possesses an antitumor activity. This enzymeis used for treating acute lymphoblastic leukemia (ALL). The aim of this study was to clone of the corresponding gene without signal peptide and also without first methionine as well as to express the gene in cytoplasm. Materials and Methods: The L-Asparaginase gene was isolated by PCR from E. coli K12 strain and cloned into engineered expression vector pET32. Sequencing and evaluation of gene expression were done by routine procedure. Methionine amino peptidase containing recombinant plasmid was purified and transferred into recombinant asparaginase producing bacterium by heat shock method. Results: The majority of L-asparaginase was expressed in cytoplasm. Based on high expression of methionine amino peptidase enzyme in E. coli Origami, it was expected that the first methionine of L-asparaginase has been removed efficiently. Conclusion: According to the achieved results, the recombinant bacterium with extensive ability to express cytoplasmic recombinant L-asparaginase is an ideal candidate for industrial production of L-asparaginase.
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