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Updated: Apr 29, 2026
Expression of Recombinant Proteins in the Methylotrophic Yeast Pichia pastoris
Published on: February 26, 2010
Pilot-scale fermentation, purification, and characterization of recombinant human Oncostatin M in Pichia pastoris
Ning Kong1, Xupeng Mu, Hongzhi Han
1Department of Biological Engineering, College of Pharmacy, Jilin University, 1163 Xin Min Street, Changchun 130021, PR China.
Abstract:
Oncostatin M (OSM) is a multifunctional cellular regulator that belongs to the IL-6 subfamily and can act on a wide variety of cells, which has potential roles in the regulation of gene activation, cell survival, proliferation and differentiation. In order to achieve the higher level yield of recombinant human Oncostatin M (rhOSM), we determined the optimal pH condition of rhOSM expressed in the methylotrophic yeast Pichia pastoris X-33 and carried out the fermentation culture of rhOSM in 80 L fermentor in a fed-batch mode. SDS-PAGE and Western blotting assays demonstrated that rhOSM was successfully expressed and secreted into the culture medium with an apparent molecular weight of 28 kDa. N-terminals were correctly processed through amino-terminal sequencing. The maximum yield of rhOSM was 280 mg/L. rhOSM was purified by phenyl Sepharose hydrophobic interaction chromatography and SP Sepharose Fast Flow cation exchange chromatography, which resulted in a final yield of purified rhOSM of 6.94 g with a recovery of 62% and a purity of 95%. The purified rhOSM had a specific growth inhibition activity of 6.26 x 10(4)RU/microg, which was commensurate with typical values (6.2 x 10(4)RU/microg) obtained with standard hOSM.
Insights
This study optimized conditions for producing recombinant human Oncostatin M (rhOSM) in Pichia pastoris, achieving a high yield of 280 mg/L. The purified rhOSM demonstrated significant growth inhibition activity, confirming its biological efficacy.
Area of Science:
- Biotechnology
- Cell Biology
- Protein Expression
Background:
- Oncostatin M (OSM), an IL-6 subfamily member, regulates gene activation, cell survival, proliferation, and differentiation.
- Optimizing recombinant human Oncostatin M (rhOSM) production is crucial for its therapeutic and research applications.
Purpose of the Study:
- To determine optimal pH for rhOSM expression in Pichia pastoris X-33.
- To achieve high-level yield of rhOSM through fed-batch fermentation.
- To purify and characterize the biological activity of the produced rhOSM.
Main Methods:
- Fed-batch fermentation of rhOSM in an 80 L fermentor using Pichia pastoris X-33.
- SDS-PAGE and Western blotting for expression analysis.
- Phenyl Sepharose and SP Sepharose Fast Flow chromatography for purification.
- Amino-terminal sequencing for N-terminal processing verification.
Main Results:
- Optimal pH conditions were determined for rhOSM expression.
- Maximum rhOSM yield reached 280 mg/L with successful secretion.
- Purified rhOSM (6.94 g, 95% purity) exhibited specific growth inhibition activity (6.26 x 10(4)RU/microg).
Conclusions:
- Pichia pastoris is an effective host for high-level rhOSM production.
- The optimized fermentation and purification process yields biologically active rhOSM.
- This work provides a scalable method for producing rhOSM for further research and potential therapeutic use.
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