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

Paper Details

Research Paper 01/09/2018
Views (594)
current_issue_feature_image
publication_file

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

S. Noor Basha, Murali K.R. Tummuru, A. Krishna Satya, Chitra Bajji, Narendranath Alluri
Int. J. Biosci. 13(3), 38-44, September 2018.
Copyright Statement: Copyright 2018; The Author(s).
License: CC BY-NC 4.0

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.

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.

Related Articles

Yield performance assessment of different mustard cultivars under field conditions

Md. Khan Jahan Ali, Md. Moshiur Rahman, Kamrun Nahar, Sharmin Ara Jannat, Mst. Khadija Khatun, Sushan Chowhan, Md. Habibur Rahman, Int. J. Biosci. 27(2), 277-282, August 2025.

Species composition of xylomicobiota of some woody plants distributed in Azerbaijan

K. F. Bakhshaliyeva, V. Y. Hasanova, N. R. Namazov, B. N. Aliyeva, S. C. Garayeva, S. E. Nagiyeva, P. Z. Muradov, Int. J. Biosci. 27(2), 267-276, August 2025.

Perception of producers and processors on sorghum diversity in the context of climate change in center and Northern Benin

Parfait Segla Alohoutade1,2,3, Alphonse Sako Avocefohoun*1,2,3, Sènan Vodouhe2, Mohamed Kanazoe1,2,3, Nicodème Chabi1, Lamine Said Baba-Moussa3, Int. J. Biosci. 27(2), 256-266, August 2025.

Implications of aberrant glycosylation on age-related disease progression

Tahmid Ahmad Patwary, Mukramur Rahman, Md. Nafis Fuad Prottoy, Sayad Md. Didarul Alam, Int. J. Biosci. 27(2), 243-255, August 2025.

Epizootic status and eradication of parasitic diseases in brown bears transferred to the rehabilitation center in Azerbaijan

Siala İ. Rustamova, Aygun A. Azizova, Gular R. Mammadova, Ramin S. Mammadov, Int. J. Biosci. 27(2), 236-242, August 2025.

Cytotoxic and apoptotic effects of Annona squamosa (Atis) crude leaf extract against A549 (Human Lung Adenocarcinoma) cell line

Ashton U. Lim, Noricel U. Garcia, Alkauzar H. Tantong, Int. J. Biosci. 27(2), 226-235, August 2025.

Perceptions of stakeholders towards sheep and goat dairy products in Benin

Yvette Adje, Philippe Sessou, Aretas Tonouhewa, Paulin Azokpota, Lamine Baba-Moussa, Souaïbou Farougou, Int. J. Biosci. 27(2), 211-225, August 2025.

Medicinal plants sold in Daloa markets: Traditional knowledge and Public health issues

Kouakou Yao Bertin, Kouakou Assoman Serge Alain, Kouame Yao Anicet Gervais, Malan Djah François, Bakayoko Adama, Int. J. Biosci. 27(2), 200-210, August 2025.