Expression and analysis of the recombinant human interleukin-21 (rhIL-21) in Pichia pastoris.
- Author:
Dong LI
1
;
Huiqing YU
;
Rongfen HUO
;
Jianquan CHEN
;
Guoxiang CHENG
Author Information
1. School of Life Science and Technology, Tongji University, Shanghai 200092, China.
- Publication Type:Journal Article
- MeSH:
Electroporation;
Genetic Vectors;
genetics;
Humans;
Interleukins;
biosynthesis;
genetics;
Pichia;
genetics;
metabolism;
Recombinant Proteins;
analysis;
biosynthesis;
genetics
- From:
Chinese Journal of Biotechnology
2009;25(11):1711-1717
- CountryChina
- Language:Chinese
-
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.
