Membrane proteins are an essential part of the machinery of life. They connect the interior and exterior of cells, play an important role in cell signaling and are responsible for the influx and efflux of nutrients and metabolites. For their structural and functional analysis high yields of correctly folded and modified protein are needed. Insect cells, such as Sf9 cells, have been one of the major expression hosts for eukaryotic membrane proteins in structural investigations during the last decade, as they are easier to handle than mammalian cells and provide more natural posttranslational modifications than microbial systems. Here we describe general techniques for establishing and maintaining insect cell cultures, the generation and amplification of recombinant baculovirus stocks using the flashBAC™ or Bac-to-Bac™ systems, membrane protein production, as well as the production of membrane preparations for extraction and purification experiments.
|Title of host publication||Methods in Molecular Biology|
|Number of pages||18|
|Publication status||Published - 1 Jul 2022|
|Name||Methods in Molecular Biology|
Bibliographical noteFunding Information:
We are grateful for funding from the European Union’s Horizon 2020 research and innovation programme under Marie Sklodowska-Curie grant agreement No. 847419 (MemTrain) as well as the ERACoBioTech MeMBrane project and BBSRC (BB/R02152X/1) to A.D.G, A.J.R, and R.M.B.
- Genetic Vectors
- Membrane Proteins/genetics
- Protein Processing, Post-Translational
- Recombinant Proteins/genetics
- Sf9 Cells
- Recombinant baculovirus
- Sf9 insect cells
- Membrane protein production