Ganoderma lucidum immunomodulatory protein(Lz-8) expressed in Pichia pastoris and the identification of immunocompetence

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 mg/mL. However, no aggregation was seen at concentration greater than 50 mg/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.