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Bridging the gap: A GFP-based strategy for overexpression and purification of membrane proteins with intra and extracellular C-termini by Jennifer M. Hsieh; Gabriel Mercado Besserer; M. Gregor Madej; Ha-Quyen Bui; Seunghyug Kwon; Jeff Abramson is a Biochemistry, Genetics and Molecular Biology article available to read on EtoBox.
What is Bridging the gap: A GFP-based strategy for overexpression and purification of membrane proteins with intra and extracellular C-termini about?
## Abstract Low expression and instability during isolation are major obstacles preventing adequate structure‐function characterization of membrane proteins (MPs). To increase the likelihood of generating large quantities of protein, C‐terminally fused green fluorescent protein (GFP) is commonly used as a reporter for monitoring expression and evaluating purification. This technique has mainly been restricted to MPs with intracellular C‐termini (C~in~) due to GFP's inability to fluoresce in the __Escherichia coli__ periplasm. With the aid of Glycophorin A, a single transmembrane spanning protein, we developed a method to convert MPs with extracellular C‐termini (C~out~) to C~in~ ones providing a conduit for implementing GFP reporting. We tested this method on eleven MPs with predicted C~out~ topology resulting in high level expression. For nine of the eleven MPs, a stable, monodisperse protein‐detergent complex was identified using an extended fluorescence‐detection size exclusion chromatography procedure that monitors protein stability over time, a critical parameter affecting the success of structure‐function studies. Five MPs were successfully cleaved from the GFP tag by site‐sp
Who reads Bridging the gap: A GFP-based strategy for overexpression and purification of membrane proteins with intra and extracellular C-termini?
It is typically read by researchers, students, and practitioners in Biochemistry, Genetics and Molecular Biology.
- Author
- Jennifer M. Hsieh; Gabriel Mercado Besserer; M. Gregor Madej; Ha-Quyen Bui; Seunghyug Kwon; Jeff Abramson
- Publisher
- Cold Spring Harbor Laboratory Press; Wiley (John Wiley & Sons); Wiley-Blackwell; Wiley (ISSN 0961-8368)
- Published
- 2010
- Language
- EN
- Field
- Biochemistry, Genetics and Molecular Biology (Life Sciences)