Membrane Protein Expression in Insect Cells Using the Baculovirus Expression Vector System.

Abstract

Integral membrane proteins have a critical role in fundamental biological processes; they are major drug targets and therefore of high research interest. Recombinant protein production is the first step in the protein tool generation for biochemical and biophysical studies. Here, we provide simplified protocols that facilitate the generation of high-quality virus and initial expression analysis for integral membrane protein targets utilizing the baculovirus-mediated expression system in insect cells. The protocol steps include generation of viruses, virus quality control, and initial expression trials utilizing standard commercial baculovirus vector systems and are exemplified for G protein-coupled receptor targets. The viral quality, quantity, and recombinant protein expression are evaluated by microscopy, flow cytometry, fluorimetry, and SDS-PAGE, using either covalently fused fluorescent proteins or co-expressed fluorescence markers. Moreover, integral membrane protein expression levels, approximate molecular mass, and stability can be evaluated from small-scale expression and purification trials.