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Related Experiment Video

Updated: Jul 10, 2026

Expression of Recombinant Proteins in the Methylotrophic Yeast Pichia pastoris
09:46

Expression of Recombinant Proteins in the Methylotrophic Yeast Pichia pastoris

Published on: February 25, 2010

Decrease of hirudin degradation by deleting the KEX1 gene in recombinant Pichia pastoris.

Zhenhua Ni1, Xiangshan Zhou, Xueqian Sun

  • 1State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, People's Republic of China.

Yeast (Chichester, England)
|November 2, 2007
PubMed
Summary

Deleting the KEX1 gene in Pichia pastoris significantly reduced hirudin degradation, increasing intact hirudin production. This genetic modification enhances recombinant hirudin yield for potential therapeutic applications.

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Area of Science:

  • Biotechnology
  • Molecular Biology
  • Biochemistry

Background:

  • Recombinant hirudin production in Pichia pastoris faces challenges with severe hirudin degradation.
  • Previous engineering efforts to improve cell viability and reduce proteinase release had limited success in preventing degradation.

Purpose of the Study:

  • To reduce hirudin degradation in Pichia pastoris by deleting the KEX1 gene.
  • To enhance the yield of intact hirudin Hir65 through genetic modification.

Main Methods:

  • Utilized a pop-in/pop-out strategy to delete the KEX1 gene in P. pastoris GS115Hir.
  • Replaced the ARG4 auxotrophic marker with the sh ble resistant marker during gene deletion.
  • Quantified intact hirudin Hir65 percentage in total hirudin under methanol fermentation conditions.

Main Results:

  • Hirudin degradation was significantly decreased in the GS115HirDeltakex1 strain compared to the undeleted strain.
  • The percentage of intact hirudin Hir65 remained high (90% initially, >62% later) in the engineered strain, versus ~40% in the control.
  • Maximal intact hirudin Hir65 concentration reached 2.4 g/l in GS115HirDeltakex1, significantly higher than 1.1 g/l in GS115Hir.

Conclusions:

  • Deletion of the KEX1 gene is an effective strategy to minimize hirudin degradation in Pichia pastoris.
  • The engineered strain GS115HirDeltakex1 demonstrates substantially improved production of intact hirudin Hir65.
  • This approach offers a promising method for increasing the yield of functional recombinant hirudin.