Squalene synthase (SS) is the key precursor and first committed enzyme of the sterol biosynthesis pathway. In a previous work, SS has been identified as one of the immunogenic proteins that could be a potential diagnostic candidate for the pathogenic fungus Candida tropicalis. In this study, SS from C. tropicalis was cloned and expressed as recombinant protein in Pichia pastoris to investigate its reactivity with serum antibodies. ERG9 gene that encodes for SS was amplified by PCR and cloned in-frame into pPICZB expression vector. The recombinant construct was then transformed into P. pastoris GS115 host strain. Expression of the recombinant protein was confirmed by SDS-PAGE and Western blot analysis using anti-His tag probe. Optimal protein production was achieved by cultivating the culture with 1.0% methanol for 72h. The recombinant protein was purified to approximately 97% pure in a single step immobilized metal affinity chromatography with a yield of 70.3%. Besides, the purified protein exhibited specific reactivity with immune sera on Western blot. This is the first report on heterologous expression of antigenic SS from C. tropicalis in P. pastoris which can be exploited for large-scale production and further research. The results also suggested that the protein might be of great value as antigen candidate for serodiagnosis of Candida infection.
Anacardium occidentale belongs to the Anacardiaceae family. It had been scientifically proven to have antihypercholesterolemia effect in high cholesterol diet induced animal laboratory study. However there is no study regarding the mechanisms involves in cholesterol reducing effect by A. occidentale leaves extract. In this study, cytotoxic assessment and anti-cholesterol activity of A. occidentale leaves aqueous extract (AOE) were investigated. Cytotoxic study was performed by exposing hepatoma cell (Hep G2) towards AOE with concentration ranging from 0.002 to 20 mg/mL for 24 h. Anacardium occidentale extract was found to be not toxic to the cell. Then, the highest and not toxic AOE concentrations (20, 10, 5 and 2.5 mg/mL) were selected for anti-cholesterol study. The ability of AOE to reduce cholesterol in cell culture experiment was carried out by pretreating Hep G2 with selected concentrations of AOE in 6-well plate before the cell was exposed to low density lipoprotein (LDL). The concentration of farnesyl-diphosphate farnesyltransferase (FDFT1), apolipoprotein A1 (Apo A1), lecithin-cholesterol acyltransferase (LCAT), low density lipoprotein receptor (LDL R), scavenger receptor B1 (SR-B1), ATP binding cassette transporter A1 (ABCA-1) and hepatic lipase (HL) were determined from the 6-well plate media. The results showed that AOE did not significantly increase the concentration of LDLR. However, AOE significantly increased the concentration of FDFT1, APO A1, LCAT, SRB-1, ABCA-1 and HL. The HMGR activity experiment showed that all selected AOE concentrations cannot significantly reduce the HMGR enzyme activity. These findings suggested that AOE may involve in reverse cholesterol transport process to reduce cholesterol metabolism in Hep G2 cell.