Abstract
Identifying membrane proteins that can be produced and isolated in homogenous form in detergent is a lengthy trial-and-error process that can be facilitated by fluorescence-based screening approaches. We describe (1) the strategy and protocol of cloning by homologous recombination, (2) whole-cell and in-gel fluorescence measurements to estimate GLUT-GFP fusion protein yields, (3) use of size-exclusion chromatography monitored by fluorescence (FSEC) for assessing the homogeneity of the GLUT-GFP fusion proteins, and (4) the protocol for large-scale production and purification of the Bos taurus GLUT5 construct that enabled its crystal structure determination.
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References
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Verdon, G., Kang, H.J., Drew, D. (2018). Screening and Scale-Up of GLUT Transporter Constructs Suitable for Biochemical and Structural Studies. In: Lindkvist-Petersson, K., Hansen, J. (eds) Glucose Transport. Methods in Molecular Biology, vol 1713. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7507-5_3
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DOI: https://doi.org/10.1007/978-1-4939-7507-5_3
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