The cloning and expression platform is focused on the rapid cloning and test expression of membrane protein constructs using the pOPIN vector system1,2. The pOPIN vector system was developed by PPUK (formerly OPPF) and enables rapid screening in E. coli, insect and mammalian cells. Cloning is not limited to the pOPIN system and other commonly used vectors include pWaldo-GFP3 are used as well as vectors for the production of membrane proteins in yeast.
To confirm expression of a membrane protein of interest we track green fluorescent protein (GFP) fluorescence during expression (where appropriate) and confirm that the membrane protein is produced using a quick IMAC pull-down screen that is analysed by SDS-PAGE and F-SEC4.
(A) Protein tagging options with the pOPIN vector system. (B) Tracking GFP fluorescence under different expression conditions in HEK293 for a membrane transporter protein. (C) SDS-PAGE from small-scale test expression and purification of a His tagged membrane protein. (D) Corresponding size exclusion profiles for a selection of constructs.
1. Berrow, N.S.; Alderton, D.; Sainsbury, S.; Nettleship, J.; Assenberg, R.; Rahman, N.; Stuart, D.I.; Owens, R.J. A versatile ligation-independent cloning method suitable for high-throughput expression screening applications. Nucleic Acids Research 2007, 35, e45-e45, doi:10.1093/nar/gkm047.
2. Bird, L.E.; Rada, H.; Flanagan, J.; Diprose, J.M.; Gilbert, R.J.C.; Owens, R.J. Application of In-Fusion™ Cloning for the Parallel Construction of E. coli Expression Vectors. In DNA Cloning and Assembly Methods, Valla, S., Lale, R., Eds. Humana Press: Totowa, NJ, 2014; 10.1007/978-1-62703-764-8_15pp. 209-234.
3. Drew, D.E.; von Heijne, G.; Nordlund, P.; de Gier, J.W. Green fluorescent protein as an indicator to monitor membrane protein overexpression in Escherichia coli. FEBS Lett 2001, 507, 220-224.
4. Birch, J.; Cheruvara, H.; Gamage, N.; Harrison, P.J.; Lithgo, R.; Quigley, A. Changes in Membrane Protein Structural Biology. Biology 2020, 9, 401.
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