Talaromyces marneffei is an intracellular dimorphic fungus that causes a fatal disseminated disease in human immune-deficient virus-infected patients. The factors that affect the pathogenicity of this fungus remain unclear. Superoxide dismutase has been shown to contribute to the virulence of many human-pathogenic fungi through its ability to neutralize toxic levels of reactive oxygen species generated by the host.  In this study, we constructed the recombinant Cu, Zn superoxide dismutase clone from T. marneffei in Pichia pastoris to determine for specific enzyme activity and Cu, Zn SOD overexpressed yeast was tested for resistance to the oxidative stress conditions. 

Full-length sodA gene encoding Cu, Zn SOD of T. marneffei, was amplified then cloned into pPICzαB plasmid and transformed into TOP10 E. coli competent cells. The positive clone was amplified and linearized with the MssI restriction enzyme. The fragments were transformed into P. pastoris X-33 competent yeast cells by electroporation and selected  the positive recombinant yeast cells with media containing zeocin™. The positive clones were then checked with PCR and sequencing. The selected clone was induced for recombinant protein expression. The recombinant Cu, Zn SOD was purified and enzymatic activity was examined by Native-PAGE activity. Moreover, the antioxidant property of Cu, Zn SOD recombinant yeast was determined by culture in heat shock condition, H2O2, paraquat or menadione.

An approximate molecular mass 20 kDa of secreted recombinant Cu, Zn SOD was detected by Coomassie blue staining-SDS-PAGE and his-tag was detected by western blot with Ni-NTA. The results from Native-PAGE revealed that the purified Cu, Zn SOD recombinant protein had an enzymatic activity that was similar to the standard enzyme. The sodA-pPICzαB recombinant yeast appeared to be more resistance to oxidative stress conditions than pPICzαB recombinant yeast. 
This is the preliminary study for expression and enzyme activity determination of recombinant Cu, Zn SOD protein from T. marneffei. The further study will focus on protein characterization of the immunogenic and virulence properties.

Expression of Cu, Zn superoxide dismutase recombinant protein from Talaromyces marneffei in Pichia pastoris and its resistance to oxidative stress