Location: Pest Management and Biocontrol ResearchTitle: Transient expression and cellular localization of recombinant proteins in cultured insect cells Author
Submitted to: Journal of Visualized Experiments
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/13/2017
Publication Date: 4/20/2017
Citation: Fabrick, J.A., Hull, J.J. 2017. Transient expression and cellular localization of recombinant proteins in cultured insect cells. Journal of Visualized Experiments. 122:e55756. doi: 10.3791/55756.
Interpretive Summary: Large-scale sequencing of DNA from many different organisms is quickly outpacing our ability to assign protein function to such genes. Hence, systems are needed to discern protein function, protein interactions, and to characterize subcellular localization. The production of foreign proteins in cellular systems using DNA isolated from organisms that differ from the cultured cells, represents an important tool for high-throughput studies of protein function and subcellular localization. Here, ARS scientists at Maricopa, AZ provide a standard protocol for generating carrier DNA molecules that allow for the synthesis of proteins of interest within commercially available insect cell cultures. These proteins are fused with proteins that naturally fluoresce and thereby provide means of detection within cells. Namely, the cellular localization of two Bemisia tabaci aquaporin water channel proteins were validated using two subcellular marker proteins with known subcellular targeting. These results show the potential for using insect cell expression systems for enhanced elucidation of cellular trafficking of proteins.
Technical Abstract: Heterologous protein expression systems are used for production of recombinant proteins, interpretation of cellular trafficking/localization, and for the determination of biochemical function of proteins at the sub-organismal level. Although baculovirus expression systems are increasingly used for protein production in numerous biotechnological, pharmaceutical and industrial applications, non-lytic systems that do not involve viral infection, have clear benefits, but are often over looked and underutilized. Here, we describe a method for generating non-lytic expression vectors and transient recombinant protein expression. We show that this protocol allows for efficient cellular localization of recombinant proteins and can be used to rapidly discern protein trafficking within the cell. We show expression of four recombinant proteins in a commercially available insect cell line, including two aquaporin proteins from the insect, Bemisia tabaci, as well as subcellular marker proteins specific for the cell plasma membrane and for intracellular lysosomes. All recombinant proteins were produced as chimeras with fluorescent protein markers at their carboxyl-termini, which allows for direct detection of the recombinant proteins. Double-transfection of cells with plasmids harboring constructs for the genes of interest and a known subcellular marker allows for live-cell imaging and improved validation of cellular protein localization.