International network for natural sciences – research journal
  • mendeley icon
  • linkedin icon
  • google plus icon
  • twitter icon
  • google scholar icon
  • facebook icon

Cloning, expression and purification of L-asparginase II from Escherichia coli in E. coli BL21DE3

By: S. Noor Basha, Murali K.R. Tummuru, A. Krishna Satya, Chitra Bajji, Narendranath Alluri

Key Words: L-Asparagnase II, Escherichia coli, Scale up, Sepharose, MALDI-TOF.

Int. J. Biosci. 13(3), 38-44, September 2018.

DOI: http://dx.doi.org/10.12692/ijb/13.3.38-44

Certification: ijb 2018 0151 [Generate Certificate]

Abstract

L-Asparagnase II (L-ASP) is a chemotherapeutic enzyme catalyses hydrolysis of asparagine into aspartate and ammonia, a key mechanism for tumour cells. L-ASP widely used for the treatment of acute lymphoblastic leukaemia and has commercial value. In this scenario, present study was aimed to sale up of recombinant L-ASP from Escherichia coli. L-ASP gene was cloned in pET 21a vector and was expressed in E. coli BL21DE3.The pH (7.2) and temperature (37°C) were optimized in shake flask, were maintained during scale up (3L and 30L) and the expression was recorded. Expressed L-ASP was captured by Diethyl amino ethyl sepharose, polished on quaternary ammonium sepharoseion exchange chromatography and purity was found to be 99.24%. The estimated yield calculated to be 1.5gm/Land the enzyme assay of purified enzyme was assayed to be 450 IU. Molecular weight of L-ASP monomer was determined by Matrix Assisted Laser Desorption Ionization-Time of Flight (MALDI-TOF) and L-ASP tetramer was estimated to be 138.92kilo Daltons. The present study concluding that the developed process might throw insights in the scale up of at industrial level for high production.

| Views 90 |

Cloning, expression and purification of L-asparginase II from Escherichia coli in E. coli BL21DE3

Aghaeepoor M, Mozafari S, Shahraki MK, Tabandeh F, Bambai B. 2011. High level of extracellular fermentation and alternative purification of Escherichia coli asparaginase II. Biharean Biologist 5,96-101.

Aghaiypour K, Wlodawer A, Lubkowski J. 2001. Structural basis for the activity and substrate specificity of Erwinia chrysanthemi L-asparaginase. Biochemistry40, 5655-5664.

Ali U, Naveed M, Ullah A, Ali K, Shah SA, Fahad S, Mumtz AS. 2016.L-asparaginase as a critical component to combat Acute Lymphoblastic Leukaemia (ALL): A novel approach to target ALL. European Journal of Pharmacology  771, 199-210.

Avramis VI, Panosyan EH. 2005. Pharmacokinetic/pharmacodynamics relationships of asparaginase formulations: the past, the present and recommendations for the future. Clinical Pharmacokinetics 44. 367-393.

Cammack KA, Marlborough DI, Miller DS.1972. Physical properties and subunit structure of L-asparaginase isolated from Erwinia carotovora. Biochemical Journal 126, 361-379.

Ebrahiminezhad A, Rasoul-Amini S, Ghasemi Y. 2011. L-Asparaginase production by moderate halophilic bacteria isolated from Maharloo salt lake. Indian Journal of Microbiology 51, 307-311.

Gladilina YA, Sokolov NN, Krasotkina JV. 2009. Cloning, expression and purification of Helicobacter pylori L-asparaginase. Biochemistry (Moscow), Supplement Series B3, 89-91.

Jayam DG, Kannan S. 2014. Screening and Characterization of L-Asparaginase Producing Streptomyces Isolated From Soil Samples of Periyar Lake, Kumily. Bioscience Discovery 5, 50-54.

Khushoo A, Pal Y, Singh BN, Mukherjee KJ. 2004. Extracellular expression and single step purification of recombinant Escherichia coli L-asparaginase II. Protein Expression and Purification 38, 29-36.

Kishore V, Nishita KP, Manonmani HK. 2015. Cloning, expression and characterization of L-asparaginase from Pseudomonas fluorescens for large scale production in E. coli BL21. 3 Biotech 5, 975-981.

Krasotkina J, Borisova AA, Gervaziev YV, Nikolay N. 2004.  Onestep purification and kinetic properties of the recombinant L’ Asparaginase from Erwinia carotovora. Biotechnology and Applied Biochemistry 39, 15-21.

Lee SM, Wroble MH, Ross JT. 1989. L-asparaginase from Erwinia carotovora. An improved recovery and purification process using affinity chromatography. Applied Biochemistry and  Biotechnology 22, 1-11.

Mitra AP, Fereshteh E, Bahram K, Joan J. 2017. Cloning, optimization of induction conditions and purification of Mycobacterium tuberculosis Rv1733c protein expressed in Escherichia coli.  Iranian Journal of Microbiology 9, 64-73.

Pourhossein M, Korbekandi H. 2014. Cloning, expression, purification and characterisation of Erwinia carotovora L-asparaginase in Escherichia coli. Advanced Biomedical Research 3, 1-6.

Raymond S, Weintraub L. 1959. Acrylamide gel as a supporting medium for zone electrophoresis. Science 130, 711.

Saeeda CH, Hadeer A, Hadeer S, Amira E, Amany El, Aida F, Ahmed H, Farid A. 2018. Molecular cloning, structural modeling and production of recombinant Aspergillus terreus l. asparaginase in Escherichia coli. International Journal of Biological Macromolecules 106, 1041-1051.

Sambrook J, Frisch E, Maniatis T. 1989. Molecular Cloning: A Laboratory Manual. 2nd Ed., New York: Cold Spring Harbor Laboratory Press., 134.

Yong Z, Xin Z, Dai-Lin Y , Bu S, Jun-Tao F, Li-Rong Han, Xing Z. 2017. Optimization of fermentation conditions and bench-scale for improvement of a novel glycoprotein GP-1 production by Streptomyces kanasenisi ZX01. Molecules 23, 1-11.

Zuo S, Xue D, Zhang T, Jaing B, Mu W. 2014. Biochemical characterization of an extremely thermostable L-asparaginase from Thermococcus gammatolerans EJ3.  Journal of  Molecular Catalysis B 109, 122-129.

S. Noor Basha, Murali K.R. Tummuru, A. Krishna Satya, Chitra Bajji, Narendranath Alluri.
Cloning, expression and purification of L-asparginase II from Escherichia coli in E. coli BL21DE3.
Int. J. Biosci. 13(3), 38-44, September 2018.
https://innspub.net/ijb/cloning-expression-purification-l-asparginase-ii-escherichia-coli-e-coli-bl21de3/
Copyright © 2018
By Authors and International Network for
Natural Sciences (INNSPUB)
https://innspub.net
brand
innspub logo
english language editing
  • CALL FOR PAPERS
    CALL FOR PAPERS
    Publish Your Article
  • CALL FOR PAPERS
    CALL FOR PAPERS
    Submit Your Article
INNSPUB on FB
Email Update