Engineering folding mechanism through Hsp70 and Hsp40 chaperones for enhancing the production of recombinant human interferon gamma (rhIFN-γ) in Pichia pastoris cell factory

Pichia pastoris is a well-known host for the production of heterologous proteins, but the incorporation of a foreign gene may lead to metabolic burden and sub optimal folding of proteins in the endoplasmic reticulum (ER) and cytoplasm, ultimately resulting in reduced protein secretion. The Hsp70 and Hsp40 chaperone families in the cytoplasm/ER are required to ensure proper folding and translocation of heterologous proteins. In the present study, we have cloned a gene encoding human interferon gamma in Pichia pastoris (GS115) resulting in a very low expression of recombinant human interferon gamma (rhIFN-γ). To evaluate the role of chaperones in protein production we have co-expressed SEC63P, YDJ1P, SSA1P, KAR2P and PDI genes from Saccharomyces cerevisiae and Pichia pastoris (X-33) respectively. The introduction of individual chaperones viz., Ydj1p, PDI and Ssa1p have enhanced rhIFN-γ production about 4 folds, while the synergistic effect of Kar2p + PDI has shown about 6-fold enhancement in rhIFN-γ production. The batch reactor kinetics with complex medium showed that the product is growth associated and maximum production yield of 1.98 mg L−1 was observed at a 72 hr time interval. To evaluate the efficacy of rhIFN-γ on an anti-proliferative property, we have treated oral cancer cell lines with different concentration of rhIFN-γ and found inhibition of cell proliferation up to 25% at 0.5 µg concentration.