title: Expression and purification of active plant diphosphonucleotide phosphatase/phosphodiesterase from baculovirus-infected insect cells.
abstract: We have previously purified and characterized a diphosphonucleotide phosphatase/phosphodiesterase (PPD1) from yellow lupin seeds. This report describes an efficient strategy for overexpression in baculovirus-infected Spodoptera frugiperda Sf9 cells and purification of a functional PPD1 enzyme. We tested six variants of recombinant PPD1, differing in secretion peptides, fusion tags, and promoters. The highest expression level of the active PPD1 was achieved when the native signal peptide and the C-terminal V5-6His tag were attached. This recombinant protein was secreted at very high level (18.4 mg/L) to serum-free medium. Single-step purification procedure using metal affinity chromatography resulted in the homogeneous PPD1. The overexpressed protein showed enzymatic activity comparable to the native enzyme isolated previously from plant material. We showed that PPD1, which belongs to purple acid phosphatase family, formed tetrameric structure, which is non-typical for this group of enzymes.
authors.affiliation: Laboratory of Biochemistry, Institute of Biochemistry and Molecular Biology, Wroclaw University, Wroclaw, Poland. olczak@bf.uni.wroc.pl
authors.forename: Mariusz
authors.initials: M
authors.lastname: Olczak
authors.forename: Teresa
authors.initials: T
authors.lastname: Olczak
mesh_terms: D000818:Animals; D016367:Baculoviridae; D015153:Blotting, Western; D002478:Cells, Cultured / Q000821:virology; D003001:Cloning, Molecular; D003470:Culture Media; D004591:Electrophoresis, Polyacrylamide Gel; D005822:Genetic Vectors; D031297:Lupinus / Q000201:enzymology*; D010727:Phosphoric Diester Hydrolases / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D018515:Plant Leaves / Q000201:enzymology; D011755:Pyrophosphatases / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D018411:Spodoptera / Q000821:virology; D013997:Time Factors
chemical_list: D003470:Culture Media; D010727:Phosphoric Diester Hydrolases; D011755:Pyrophosphatases; C457079:diphosphonucleotide phosphatase-phosphodiesterase 1