Internal ribosome entry site mediates protein synthesis in yeast Pichia pastoris

The imitation of translation, as mediated by internal ribosome entry sites, has not yet been reported in Pichia pastoris. An IRES element from Saccharomyces cerevisiae was demonstrated to direct the translation of a dicistronic mRNA in P. pastoris. The 5′-untranslated region of GPR1 mRNA, termed GPR, was cloned into a dual reporter construct containing an upstream Rhizomucor miehei lipase (RML) and a downstream β-galactosidase gene (lacZ) from Escherichia coli BL21. After being transformed into P. pastoris, the RML gene and lacZ were simultaneously expressed. The possibility of DNA rearrangement, spurious splicing, or cryptic promoter in the GPR sequence were eliminated, indicating that expression of a second ORF was IRES-dependent. These findings strongly suggested that the IRES-dependent translation initiation mechanism is conserved in P. pastoris and provides a useful means to express multiple genes simultaneously.