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

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Research Paper 01/09/2018
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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.
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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.


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