1. Membrane Protein Expression in Insect Cells Using the Baculovirus Expression Vector System.
- Author
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Boivineau J, Haffke M, and Jaakola VP
- Subjects
- Animals, Baculoviridae growth & development, Bioreactors, Cell Culture Techniques methods, Cell Line, Gene Expression Regulation, Viral, Green Fluorescent Proteins genetics, Green Fluorescent Proteins isolation & purification, Green Fluorescent Proteins metabolism, Membrane Proteins isolation & purification, Membrane Proteins metabolism, Recombinant Proteins genetics, Recombinant Proteins isolation & purification, Recombinant Proteins metabolism, Spodoptera genetics, Spodoptera growth & development, Spodoptera metabolism, Transduction, Genetic methods, Transfection methods, Baculoviridae genetics, Cloning, Molecular methods, Genetic Vectors genetics, Genetic Vectors metabolism, Membrane Proteins genetics, Spodoptera cytology
- 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.
- Published
- 2020
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