Ganoderma lucidum immunomodulatory protein(Lz-8) expressed in Pichia pastoris and the identification of immunocompetence.
- Author:
Chongyang LIANG
1
;
Shuqin ZHANG
;
Zhiyi LIU
;
Fei SUN
Author Information
1. Department of Cytobiology, Institute of Frontier Medical Science, Jilin University, Changchun 130021, China.
- Publication Type:Journal Article
- MeSH:
Fungal Proteins;
biosynthesis;
genetics;
immunology;
Immunocompetence;
immunology;
Macrophages;
immunology;
Phagocytosis;
drug effects;
Pichia;
genetics;
metabolism;
Recombinant Proteins;
biosynthesis;
genetics;
immunology
- From:
Chinese Journal of Biotechnology
2009;25(3):441-447
- CountryChina
- Language:English
-
Abstract:
Fungi immunoregulatory proteins family is effective in immunological regulation and anti-tumor. We used Pichia pastoris expression system for recombinant expression of Lz-8, an immunomodulatory protein isolated from fruiting body of Ganoderma lucidum. The Gs115 (mut+) strains of P. pastoris was used as host cells. PCR and sequencing of DNA showed that Lz-8 cDNA was successfully integrated into the P. pastoris genome. Electrophoresis (SDS-PAGE), matrix assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF-MS) and immunological techniques were used to identify recombinant Lz-8 (rLz-8). Lz-8 expressed in Escherichia coli, the Pichia system requires further optimization to obtain more active fungi immunomodulatory protein. Lz-8 was expressed in P. pastoris successfully, and polyacrylamide gel electrophoresis in the presence of SDS-PAGE gave a single band with an apparent Mr=14,000 D. MALDI-TOF-MS also showed that molecular weight of rLz-8 was 12,722 D. Aggregation was observed from sheep red blood cells in the presence of purified rLz-8 within the concentration range of 12.5-50 microg/mL. However, no aggregation was seen at concentration greater than 50 microg/mL for any type of human red blood cell. The dose at 0.5 mg/kg of rLz-8 induced macrophage cytophagocytesis, and set interferon as control at 0.5 mg/kg. These results suggested that active and stable rLz-8 was obtained in P. pastoris expression system.
