Title
Heterologous Expression and Functional Characterization of Phytaspase, a Caspase-Like Plant Protease
Author
Sharmila Narayanan, Pallab Sanpui, Lingaraj Sahoo, Siddhartha Sankar Ghosh
Year
2016
Journal
International Journal of Biological Macromolecules
Abstract
Following the cloning and expression of tobacco (Nicotiana tabacum) phytaspase gene in Escherichia coli BL21, the recombinant protease was purified by affinity chromatography for further characterization. Circular dichroism (CD) spectroscopy and in silico analysis revealed structural similarities of recombinant phytaspase with other plant serine-proteases. Molecular docking studies showed favourable binding of synthetic peptide substrate for caspase 8 (Ac-VETD-AMC) to the reactive pocket of recombinant phytaspase indicating its potential in assessing functional activity of recombinant phytaspase. In silico findings were supported by caspase 8-like activity of purified phytaspase demonstrated in vitro. The Michaelis constant (KM) and specificity constant (kcat/KM) of phytaspase for hydrolyzing Ac-VETD-AMC were found to be 1.587 μM and 4.67 × 103 M−1 min−1, respectively. Transient expression of phytaspase in epithelial adenocarcinoma cells (A549) resulted in reduced IC50 value of doxorubicin. This is the first report of functional expression of mature phytaspase in bacterial system as well as its transfection to sensitize A549 cells at lower doxorubicin concentration.
Instrument
J-815
Keywords
Circular dichroism, Secondary structure, Biochemistry