Cloning, Sequence analysis, and expression of L-Asparaginase obtained from Erwinia chrysanthemi
Subject Areas : Microbial BiotechnologyRazieh Afrasiabi 1 , Khosrow Aghaiypour 2 , Farshid Kafilzadeh 3 , Sadighe Safaviyeh 4
1 - Department of Microbiology, Jahrom Branch, Islamic Azad University, Jahrom, Iran
2 - Department of Biotecnology, Razi Vaccine and Serum Research Institute, Karaj, Iran
3 - Department of Microbiology, Jahrom Branch, Islamic Azad University, Jahrom, Iran
4 - Department of Biotecnology, Razi Vaccine and Serum Research Institute, Karaj, Iran
Keywords: SDS-PAGE, pAED4, L- asparaginase, Erwinia chrysanthemi,
Abstract :
Background and objectives: L-asparaginases enzyme is an effective drugs for treatment of lymphoblasticleukemia and Non-Hodgkin's lymphoma. Escherichia coli and Erwinia strains specially Erwinia chrysanthemi are more important producers of this enzyme. The aim of this study was survey on L-asparaginase enzyme obtained from Erwinia chrysanthemi DSM4610. Materials and methods: After bacterial genomic DNA extraction, a PCR procedure was performed with specific primers, Echr Asn F and Echr Asn R1. At the next step, the amplified DNA segment were ligated in a pTZ57R/T vector and after transformation into E. coli DH5á, the recombinant plasmids were extracted. After sequencing, the results were analyzed by DNAMAN software. By designin its expression primers, the gene was cloned in a pAED4 vector and after transformation into E.coli BL21(DE3) cells, its expression was assessed by SDS-PAGE analysis. Results: The sequence of the isolated gene from Erwinia chrysanthemi DSM4610 was different from other similar genes in Gene Bank and was assigned with the accession number: JF972567 in Gene Bank. The most expression was found in the condition of 0.5 mM of IPTG, LB broth media and 37°C. SDS-PAGE analysis showed 37.5 KD protein. Conclusion: Results revealed that the L-asparaginase enzyme obtained of Erwinia chrysanthemi has proper features for further study as an antileukemia enzyme.
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