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

Research output: Contribution to journalArticle

3 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)673-678
Number of pages6
JournalJournal of Bioscience and Bioengineering
Volume122
Issue number6
DOIs
Publication statusPublished - 2016 Dec 1

Fingerprint

Hyaluronoglucosaminidase
Pichia
Genes
Acetylcysteine
Hyaluronic acid
Acids
Molecular mass
Plasmids
Periodic Acid
DNA
Fusion reactions
Adhesion
Enzymes
Resins
Hyaluronic Acid
Proteins
Adsorption
Molecules
Inhibitory Concentration 50
Cysteine

Keywords

  • Functional expression
  • Glycoprotein
  • Human hyaluronidase
  • PH20
  • Pichia pastoris

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Applied Microbiology and Biotechnology

Cite this

Constitutive expression of recombinant human hyaluronidase PH20 by Pichia pastoris. / Chen, Kuan Jung; Sabrina, Sabrina; El-Safory, Nermeen S.; Lee, Guan Chiun; Lee, Cheng Kang.

In: Journal of Bioscience and Bioengineering, Vol. 122, No. 6, 01.12.2016, p. 673-678.

Research output: Contribution to journalArticle

Chen, Kuan Jung ; Sabrina, Sabrina ; El-Safory, Nermeen S. ; Lee, Guan Chiun ; Lee, Cheng Kang. / Constitutive expression of recombinant human hyaluronidase PH20 by Pichia pastoris. In: Journal of Bioscience and Bioengineering. 2016 ; Vol. 122, No. 6. pp. 673-678.
@article{7b5b5fb11ef74f5c9b642be858fb3f40,
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.",
keywords = "Functional expression, Glycoprotein, Human hyaluronidase, PH20, Pichia pastoris",
author = "Chen, {Kuan Jung} and Sabrina Sabrina and El-Safory, {Nermeen S.} and Lee, {Guan Chiun} and Lee, {Cheng Kang}",
year = "2016",
month = "12",
day = "1",
doi = "10.1016/j.jbiosc.2016.06.007",
language = "English",
volume = "122",
pages = "673--678",
journal = "Journal of Bioscience and Bioengineering",
issn = "1389-1723",
publisher = "Elsevier",
number = "6",

}

TY - JOUR

T1 - Constitutive expression of recombinant human hyaluronidase PH20 by Pichia pastoris

AU - Chen, Kuan Jung

AU - Sabrina, Sabrina

AU - El-Safory, Nermeen S.

AU - Lee, Guan Chiun

AU - Lee, Cheng Kang

PY - 2016/12/1

Y1 - 2016/12/1

N2 - 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.

AB - 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.

KW - Functional expression

KW - Glycoprotein

KW - Human hyaluronidase

KW - PH20

KW - Pichia pastoris

UR - http://www.scopus.com/inward/record.url?scp=84979650007&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84979650007&partnerID=8YFLogxK

U2 - 10.1016/j.jbiosc.2016.06.007

DO - 10.1016/j.jbiosc.2016.06.007

M3 - Article

C2 - 27373489

AN - SCOPUS:84979650007

VL - 122

SP - 673

EP - 678

JO - Journal of Bioscience and Bioengineering

JF - Journal of Bioscience and Bioengineering

SN - 1389-1723

IS - 6

ER -