title: Heterologous expression and functional characterization of phytaspase, a caspase-like plant protease.
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 1.587μM and 4.67×103M-1min-1, respectively. Transient expression of phytaspase in lung epithelial adenocarcinoma cells (A549) resulted in reduced IC50value 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.
authors.affiliation: Department of Biosciences & Bioengineering, Indian Institute of Technology Guwahati, Guwahati-39, Assam, India.
authors.forename: Sharmila
authors.initials: S
authors.lastname: Narayanan
authors.affiliation: Centre for Nanotechnology, Indian Institute of Technology Guwahati, Guwahati-39, Assam, India.
authors.forename: Pallab
authors.initials: P
authors.lastname: Sanpui
authors.affiliation: Department of Biosciences & Bioengineering, Indian Institute of Technology Guwahati, Guwahati-39, Assam, India.
authors.forename: Lingaraj
authors.initials: L
authors.lastname: Sahoo
authors.affiliation: Department of Biosciences & Bioengineering, Indian Institute of Technology Guwahati, Guwahati-39, Assam, India; Centre for Nanotechnology, Indian Institute of Technology Guwahati, Guwahati-39, Assam, India. Electronic address: sghosh@iitg.ernet.in.
authors.forename: Siddhartha Sankar
authors.initials: SS
authors.lastname: Ghosh
mesh_terms: D000072283:A549 Cells; D020169:Caspases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D003001:Cloning, Molecular; D004317:Doxorubicin / Q000494:pharmacology; D015870:Gene Expression; D006801:Humans; D062105:Molecular Docking Simulation; D017433:Protein Structure, Secondary; D013379:Substrate Specificity; D014026:Nicotiana / Q000201:enzymology* / Q000235:genetics
chemical_list: D004317:Doxorubicin; D020169:Caspases
keywords: Caspase; Molecular docking; Nicotiana tabacum; Phytaspase; Programmed cell death; Serine proteases