Using internal ribosome entry sites to facilitate engineering of insect cells and used in secretion proteins production

Abstract

Abstract Insect cells have been extensively used for the production of recombinant proteins. The baculovirus expression vector system (BEVS) are routinely used both in laboratories and in industries for the production of recombinant proteins. However, it has been established that secretory proteins can be produced more efficiently in the non-lytic, plasmid-based baculovirus-free insect system (BFIS) than in BEVS. A non-lytic, plasmid based, bi-cistronic insect cell expression system was developed by incorporating the internal ribosomal entry site (IRES) elements derived from Rhopalosiphum padi virus (RhPV IRES) or Perina nuda virus (PnV539 IRES). Both IRESes had been demonstrated to mediate bi-cistronic gene expression in baculovirus-infected Sf21 cell. However, only the PnV539 IRES functioned well in Sf21 cells through the plasmid-based baculovirus-free insect cell expression system. Based on this observation, we further combined the PnV539 IRES with enhanced green fluorescent protein (EGFP) along with blasticidin-resistant gene to facilitate the isolation of the stably transformed insect cells that expressed the target gene as observed by fluorescence microscopy. A pharmaceutically important secreted protein interferon-γ and secretory alkaline phosphatase (SEAP) was successfully produced in stably transformed Sf21 cells and further proved the application of the novel IRES-based bi-cistronic BFIS.