Identification, heterologous expression and detection of enzymatic activity of an asparaginase from the archaeon Thermoplasma acidophilum

Abstract

Asparaginases (ASNases) participate in the metabolism of all living organisms in the hydrolysis of free asparagines. Bacterial ASNases are used in cancer chemotherapy as they efficiently deplete amino acids. However, allergic reactions and silent inactivation represent critical limitations to their extended use. The rationale of this study was to identify, express, and characterize a plant-type L-ASNase from the archaeon Thermoplasma acidophilum as an enzyme with potentially improved characteristics. The Ta0338 orf was cloned into the pET28a(+) expression vector and overexpressed as a soluble protein with a molecular weight of 32 kDa. The quantity of recombinant L-ASNase produced in Escherichia coli was estimated as 9.68 mg/l. The purified protein showed evident autocatalytic processing of the zymogen at 4° and 37°C at physiological pH of 7.2 and clearly generated the expected alpha and beta subunits of 18 and 13 kDa, respectively. We propose that Ta-ASNase represents a potential biotechnological product for therapeutic purposes.

Keywords::

• Autocatalytic processing
• acute lymphoblastic leukemia
• plant-type L-asparaginase
• thermoplasma acidophilum

Acknowledgement

Mabel Guzmán Rodríguez and María Guadalupe Serna-Domínguez were supported by the National Council for Science and Technology (CONACYT), Mexico with the Graduate scholarship No. 263350 and Master in Science Scholarship No. 250294, respectively. Molecular graphics and analyses were performed with the UCSF-Chimera package. Chimera was developed by the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco (supported by NIGMS P41-GM103311).

Declaration of interest: The authors report no declarations of interest. The authors alone are responsible for the content and writing of the paper.