Heterologous expression and functional characterization of phytaspase, a caspase-like plant protease

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 (K_M) and specificity constant (k_cat/K_M) of phytaspase for hydrolyzing Ac-VETD-AMC were found to be 1.587μM and 4.67×10³M^-1min^-1, respectively. Transient expression of phytaspase in lung 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.

Caspase; Molecular docking; Nicotiana tabacum; Phytaspase; Programmed cell death; Serine proteases.