Related Experiment Video
Updated: Jun 15, 2026
Expression of Recombinant Proteins in the Methylotrophic Yeast Pichia pastoris
Published on: February 25, 2010
[Expression and analysis of the recombinant human interleukin-21 (rhIL-21) in Pichia pastoris]
Dong Li1, Huiqing Yu, Rongfen Huo
1School of Life Science and Technology, Tongji University, Shanghai 200092, China.
Insights
Researchers successfully produced bioactive recombinant human Interleukin-21 (rhIL-21) in Pichia pastoris. This breakthrough in recombinant protein expression advances potential applications in immunotherapy and cancer therapy.
Area of Science:
- Immunology
- Biotechnology
- Molecular Biology
Background:
- Interleukin-21 (IL-21) is a critical type I cytokine produced by CD4+ T cells, regulating immune responses.
- Efficient and scalable production of bioactive IL-21 is essential for its therapeutic applications.
Purpose of the Study:
- To express and purify biologically active recombinant human IL-21 (rhIL-21) in the methylotrophic yeast Pichia pastoris.
- To establish a foundation for further research in IL-21-based immunotherapy and cancer therapy.
Main Methods:
- Human IL-21 cDNA was amplified and cloned into the pPIC9K vector.
- The recombinant vector was transformed into Pichia pastoris strain GS115.
- Recombinant protein expression was induced with methanol, followed by purification using ion-exchange chromatography.
Main Results:
- Recombinant human IL-21 (rhIL-21) was successfully expressed and secreted into the culture supernatant.
- SDS-PAGE confirmed the molecular weight of rhIL-21 at approximately 16 kD.
- High yield (229.28 mg/L) and purity (95%) of rhIL-21 were achieved.
- Purified rhIL-21 demonstrated bioactivity by promoting human lymphocyte proliferation.
Conclusions:
- This study reports the first successful expression of bioactive rhIL-21 in Pichia pastoris.
- The developed system provides a robust platform for producing rhIL-21 for therapeutic research.
- This work supports the advancement of IL-21 in immunotherapy and cancer treatment strategies.
Abstract:
Interleukin-21 is a type I cytokine mainly produced by activated CD4+ T cells that acts as a regulator of immune system. In this work, hIL-21cDNA was amplified from human peripheral blood lymphocytes by RT-PCR, and then inserted into pPIC9K. The recombinant vector pPIC9K-hIL21cDNA was linearized by Sac I, and transformed into Pichia pastoris strain GS115 by electroporation. Transformants were selected by G418 and confirmed by PCR. The recombinant protein was expressed and secreted into the supernatant after inducing by methanol. SDS-PAGE analysis indicated the molecular weight of rhIL-21 was about 16 kD. ELISA results show that the yield of rhIL-21 reach 229.28 mg/L, rhIL-21 was purified from culture supernatants, and it was purified to about 95% purity with ion-exchange chromatography. When co-stimulate with Con A, rhIL-21 can promote the proliferation of human lymphocytes. This is the first expression of bio-active rhIL-21 in Pichia pastoris. It lays a foundation for further research in immunotherapy and cancer therapy.
