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

• Main Authors: Ashok Pandey, Carlos Ricardo Soccol, Ushasree Mrudula Vasudevan, Jalaja Vidya
• Format: Article
• Language: English
• Published: University of Zagreb 2011-01-01
• Series: Food Technology and Biotechnology
• Subjects: functional expression; L-asparaginase II; enzymatic property
• Online Access: http://hrcak.srce.hr/file/105751

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.