title: Expression and characterization of a recombinant unique acid phosphatase from kidney bean hypocotyl exhibiting chloroperoxidase activity in the yeast Pichia pastoris.
abstract: We previously purified and characterized a novel acid phosphatase (KhACP) from kidney bean hypocotyls that exhibited vanadate-dependent chloroperoxidase (V-CPO) activity. In the present study, a functional recombinant KhACP (rKhACP) was successfully produced at a high expression level by the methylotrophic yeast Pichia pastoris. The KhACP cDNA excising signal peptide sequence was subcloned into the pPICZalphaA vector and then integrated into the genome of P. pastoris strain X-33 under control of the alcohol oxidase 1 promoter. The rKhACP protein, with a molecular mass of 60 kDa on SDS-PAGE, was secreted into the culture medium as a C-terminal His-tagged fusion protein. Purification was facile using only nickel affinity chromatography. The apparent molecular mass of the purified rKhACP was estimated to be around 110 kDa by analytical gel filtration. PAGE analysis showed that rKhACP was a glycosylated dimeric enzyme, consisting of two 60-kDa subunits linked non-covalently, which was similar to the dominant form of the natural enzyme isolated from plant material. Furthermore, the rKhACP exhibited V-CPO activity when ortho-vanadate (VO4(3-)) was added to the apo enzyme, and it showed broad substrate specificity and kinetic parameters comparable to the natural enzyme. This expression system produces sufficient protein to allow us to attempt to determine the three-dimensional crystal structure, which will shed light on its unique mechanism of converting KhACP to vanadate-dependent chloroperoxidase.
authors.affiliation: Department of Food Science and Technology, Faculty of Bioindustry, Tokyo University of Agriculture, 196 Yasaka, Abashiri 099-2493, Japan.
authors.forename: Tohru
authors.initials: T
authors.lastname: Yoneyama
authors.forename: Maya
authors.initials: M
authors.lastname: Taira
authors.forename: Tomonori
authors.initials: T
authors.lastname: Suzuki
authors.forename: Masao
authors.initials: M
authors.lastname: Nakamura
authors.forename: Koichi
authors.initials: K
authors.lastname: Niwa
authors.forename: Toshihiro
authors.initials: T
authors.lastname: Watanabe
authors.forename: Tohru
authors.initials: T
authors.lastname: Ohyama
mesh_terms: D000135:Acid Phosphatase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D000595:Amino Acid Sequence; D001051:Apoenzymes / Q000378:metabolism; D015153:Blotting, Western; D002711:Chloride Peroxidase / Q000378:metabolism*; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D016681:Genome, Fungal; D006031:Glycosylation; D006639:Histidine / Q000737:chemistry; D018546:Hypocotyl / Q000201:enzymology; D007150:Immunohistochemistry; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D027805:Phaseolus / Q000201:enzymology*; D010843:Pichia / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D014638:Vanadates / Q000378:metabolism
chemical_list: D001051:Apoenzymes; D000939:Epitopes; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; D014638:Vanadates; D006639:Histidine; D002711:Chloride Peroxidase; D000135:Acid Phosphatase