Comparative analysis of the activity of two promoters in insect cells

Abstract

Insect cell expression system is widely used for the production of many thousands of recombinant proteins. Non-lytic vector-based expression provides a reliable result for investigating gene functions and molecular pathways, because baculovirus-mediated expression will lyse cells eventually. There are currently two major commercial vector providers. The pIB/V5-His vector from invitrogen uses the OpIE2 promoter and the pIEX-4 vector from novagen uses the IE1 promoter and hr5 enhancer. To compare the activity of these two promoters, we replaced the OpIE2 promoter in the pIB/V5-His vector with the hr5- IE1 promoter from pIEX-4. By measuring the activity of β-galactosidase under control of these two promoters, we found that the hr5-IE1 promoter showed about 20% higher activity than the OpIE2 promoter for both transient and stable expression. To facilitate protein expression in the secretion pathway, we cloned an AKH signal peptide downstream of the hr5-IE1 promoter. By comparing the cytosolic expression and localization of GFP, AKH-GFP and AKH-GFP-GPI, we found that the AKH signal peptide was sufficient to guide the passenger protein into the secretion pathway. Furthermore, to allow researchers to use different antibiotics for stable expression, we replaced the blasticidin resistance ORF with the zeocin resistance ORF and found that the two vectors yielded similar expression levels. Taken together, the hr5-IE1 is a stable and more effective combination to direct expression in insect cell. Vectors constructed in this study will provide a new choice for biological researchers using insect cell model systems.

Key words: Insect cells, vector, secretion, expression.