Expression, purification, and characterization of pro-phenoloxidase-activating serine protease from Spodoptera litura

• Published in: Archives of insect biochemistry and physiology 2009-10, Vol.72 (2), p.61-73
• Main Authors: Arora, Naresh, Hoque, M.E, Rajagopal, R, Sachdev, Bindiya, Bhatnagar, Raj K
• Format: Article
• Language: English
• Subjects: activation of phenoloxidase; Amino Acid Sequence; amino acid sequences; Animals; apoprotenin; Baculovirus; Cloning, Molecular; complementary DNA; Enzyme Activation; enzyme activity; enzyme inhibition; enzyme inhibitors; gene expression; Gene Expression Regulation, Enzymologic - genetics; innate immunity; Lepidoptera; melanization; molecular cloning; Molecular Sequence Data; monophenol monooxygenase; Monophenol Monooxygenase - metabolism; Phylogeny; prophenol oxidase activating enzyme 1; prophenoloxidase; prophenoloxidase activation; purification; recombinant proteins; Sequence Alignment; sequence analysis; Serine Endopeptidases - biosynthesis; Serine Endopeptidases - genetics; Serine Endopeptidases - isolation & purification; serine protease; serine proteinases; Spodoptera - enzymology; Spodoptera litura
• ISSN: 0739-4462; 1520-6327
• DOI: 10.1002/arch.20323

One of the important trigger molecules for innate immunity is a serine protease that activates zymogen phenol oxidase (PPO). Central to wound healing response is the activation of phenol oxidase zymogen. Molecular characterization of phenol oxidase has been recently reported by us. Here, we report isolation, cloning, expression, and purification of prophenol oxidase activating enzyme 1 (slppae1) from polyphagous pest, Spodoptera litura. SLPPAE1 is induced within 6 h of physical injury. The structural features of the mature polypeptide are reminiscent of other lepidopteran PPAE in having a signal peptide, propeptide, and catalytically active polypeptide. The cDNA has been expressed in Sf21 cells using baculovirus expression vector. Fractionation of expressing Sf21 cells revealed its expression in the membranes. The recombinant protein was solubilized from membranes and purified by Ni-NTA affinity chromatography. The purified enzyme is catalytically active on chromogenic substrate, activates recombinantly expressed prophenol oxidase (PPO) of S. litura, and is sensitive to inhibition by aprotenin. N-terminal sequencing of processed phenol oxidase revealed 11 kDa propeptide instead of in-silico predicted 6 kDa polypeptide.