Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli
| Autor: | Jan Lӧwe, Heather Rada, Anil Verma, Louise E. Bird, Matthew Jennions, Isabel Moraes, Raymond J. Owens, Raphael Gasper, James M. Birch |
|---|---|
| Rok vydání: | 2015 |
| Předmět: |
General Immunology and Microbiology
General Chemical Engineering General Neuroscience Ligation-independent cloning Biology medicine.disease_cause Molecular biology General Biochemistry Genetics and Molecular Biology Green fluorescent protein Transmembrane domain Membrane protein Biochemistry medicine Heterologous expression Ultracentrifuge Polyacrylamide gel electrophoresis Escherichia coli |
| Zdroj: | Journal of Visualized Experiments. |
| ISSN: | 1940-087X |
| DOI: | 10.3791/52357 |
| Popis: | The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|