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Cloning, Functional Expression and Characterization of L-Asparaginase II from E. coli MTCC 739

Jalaja Vidya1, Ushasree Mrudula Vasudevan1, Carlos Ricardo Soccol2 and Ashok Pandey1*

1Biotechnology Division, National Institute for Interdisciplinary Science and Technology (NIIST), 695 019 Trivandrum, India
2Bioprocess Engineering and Biotechnology Division, Federal University of Paraná (UFPR), P.O. Box 19011, CEP 81531-970 Curitiba, PR, Brazil

Article history:
Received September 13, 2010
Accepted January 11, 2011

Key words:
functional expression, L-asparaginase II, enzymatic property

Summary:

L-Asparaginase is an antineoplastic agent that selectively decreases the level of L-asparagine in blood and diminishes the proliferation of the cancerous cells. L-Asparaginases from Escherichia coli are widely used for clinical application because of their high substrate specificity and limited glutaminase activity. L-Asparaginase II-encoding gene ansB was isolated by excluding the native signal from E. coli MTCC 739, cloned in frame with pelB leader sequence of prokaryotic expression vector pET20b and expressed in E. coli DE3 cells. Overexpression of recombinant protein was achieved with an optimized final concentration of 10 μM of isopropyl β-D-1-thiogalactopyranoside (IPTG). The protein was expressed as soluble protein. The recombinant protein contained hexahistidine tag at C-terminus and was purified using nickel-nitrilotriacetic acid chromatography. Enzymatic properties such as optimum temperature, pH and the effect of temperature on the stability of L-asparaginase II from E. coli MTCC 739 were determined and the purified protein showed an optimum activity at 37 °C and pH=6.



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