Constitutive expression of recombinant human hyaluronidase PH20 by Pichia pastoris

Kuan Jung Chen, Sabrina Sabrina, Nermeen S. El-Safory, Guan Chiun Lee, Cheng Kang Lee

研究成果: 雜誌貢獻文章

4 引文 斯高帕斯(Scopus)

摘要

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.

原文英語
頁(從 - 到)673-678
頁數6
期刊Journal of Bioscience and Bioengineering
122
發行號6
DOIs
出版狀態已發佈 - 2016 十二月 1

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Applied Microbiology and Biotechnology

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