Ganoderma lucidum immunomodulatory protein(Lz-8) expressed in Pichia pastoris and the identification of immunocompetence.
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.
Liang
Chongyang
C
Department of Cytobiology, Institute of Frontier Medical Science, Jilin University, Changchun 130021, China.
Zhang
Shuqin
S
Liu
Zhiyi
Z
Sun
Fei
F
D005656:Fungal Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology*; D007121:Immunocompetence / Q000276:immunology*; D008264:Macrophages / Q000276:immunology; D010587:Phagocytosis / Q000187:drug effects; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*
D005656:Fungal Proteins; D011994:Recombinant Proteins; C061148:LZ-8 protein, Ganoderma lucidum