title: Constitutive expression of recombinant human hyaluronidase PH20 by Pichia pastoris.
abstract: PH20 is known as sperm adhesion molecule 1 (SPAM1) and also has hyaluronidase function to preferentially hydrolyze the glycosidic linkage of hyaluronic acid (HA). A DNA fragment containing core domain of human PH20 gene was cloned into a constitutive expression plasmid (pGAPZαC) of Pichia pastoris to produce a fusion protein with α factor signal in the N-terminus and 6 × His as well as c-Myc tags in the C-terminus. The resulting plasmid pGAPZαC-PH20 was integrated into the genome of P. pastoris strain GS115. Functional recombinant human PH20 (rHuPH20) was successfully expressed and secreted by the recombinant P. pastoris transformant. Highest hyaluronidase activity of 2 mU/mL could be obtained at 3 day in an YPD culture. After purified by phenylboronic acid resin adsorption, rHuPH20 with a specific activity of 230 mU/mg was obtained. Via periodic acid-Schiff staining and zymogram analysis, the partially purified rHuPH20 was determined to be highly glycosylated to various extents with molecular mass in the range of 100-300 kDa. The enzyme showed a maximal activity at pH 5.0 but no appreciable activity at pH ≤3 and pH ≥8. The hyaluronidase activity could be stably maintained at 4°C but lost 40% after incubating at 30°C for 4 h. Both N-acetyl cysteine and glutathione showed a half maximal inhibitory concentration (IC50) of 8 mM against rHuPH20.
authors.affiliation: Department of Chemical Engineering, National Taiwan University of Science and Technology, Taipei 106-07, Taiwan, ROC.
authors.forename: Kuan-Jung
authors.initials: KJ
authors.lastname: Chen
authors.affiliation: Department of Chemical Engineering, National Taiwan University of Science and Technology, Taipei 106-07, Taiwan, ROC.
authors.forename: Sabrina
authors.initials: S
authors.lastname: Sabrina
authors.affiliation: Department of Chemical Engineering, National Taiwan University of Science and Technology, Taipei 106-07, Taiwan, ROC.
authors.forename: Nermeen S
authors.initials: NS
authors.lastname: El-Safory
authors.affiliation: Department of Life Science, National Taiwan Normal University, Taipei, Taiwan, ROC.
authors.forename: Guan-Chiun
authors.initials: GC
authors.lastname: Lee
authors.affiliation: Department of Chemical Engineering, National Taiwan University of Science and Technology, Taipei 106-07, Taiwan, ROC. Electronic address: cklee@mail.ntust.edu.tw.
authors.forename: Cheng-Kang
authors.initials: CK
authors.lastname: Lee
mesh_terms: D015815:Cell Adhesion Molecules / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D015966:Gene Expression Regulation, Fungal; D006031:Glycosylation; D006801:Humans; D006820:Hyaluronic Acid / Q000378:metabolism; D006821:Hyaluronoglucosaminidase / Q000235:genetics* / Q000378:metabolism; D060847:Metabolic Engineering; D008970:Molecular Weight; D030781:Organisms, Genetically Modified; D010843:Pichia / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics* / Q000378:metabolism; D014162:Transfection
chemical_list: D015815:Cell Adhesion Molecules; D011994:Recombinant Proteins; D006820:Hyaluronic Acid; D006821:Hyaluronoglucosaminidase; C066852:hyaluronidase PH-20
keywords: Functional expression; Glycoprotein; Human hyaluronidase; PH20; Pichia pastoris