Membrane Protein Production and Purification from Escherichia coli and Sf9 Insect Cells
| Autor: | Ana Pavić, Joshua T Farley, Carine de Marcos Lousa, Yixin Liu, Vincent L. G. Postis, Adrian Goldman |
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| Přispěvatelé: | Postis, Vincent L. G., Goldman, Adrian, Molecular and Integrative Biosciences Research Programme, Biochemistry and Biotechnology |
| Rok vydání: | 2020 |
| Předmět: |
Bacterial expression
HIGH-LEVEL EXPRESSION SCALE-UP Sf9 Affinity chromatography medicine.disease_cause Baculovirus-infected insect cells Green fluorescent protein law.invention BACULOVIRUS DNA 03 medical and health sciences law Protein purification medicine CRYSTAL-STRUCTURE RECOMBINANT PROTEINS Escherichia coli FUSION SYSTEMS 030304 developmental biology 0303 health sciences Chemistry 030302 biochemistry & molecular biology AFFINITY-CHROMATOGRAPHY CARRIER PROTEINS Promoter In-gel fluorescence Membrane protein Biochemistry Recombinant DNA 1182 Biochemistry cell and molecular biology Protein expression VECTORS Green fluorescence protein OVEREXPRESSION |
| Zdroj: | Methods in Molecular Biology ISBN: 9781071607237 |
| Popis: | A major obstacle to studying membrane proteins by biophysical techniques is the difficulty in producing sufficient amounts of materials for functional and structural studies. To overexpress the target membrane protein heterologously, especially an eukaryotic protein, a key step is to find the optimal host expression system and perform subsequent expression optimization. In this chapter, we describe protocols for screening membrane protein production using bacterial and insect cells, solubilization screening, large-scale production, and commonly used affinity chromatography purification methods. We discuss general optimization conditions, such as promoters and tags, and describe current techniques that can be used in any laboratory without specialized expensive equipment. Especially for insect cells, GFP fusions are particularly useful for localization and in-gel fluorescence detection of the proteins on SDS-PAGE. We give detailed protocols that can be used to screen the best expression and purification conditions for membrane protein study. |
| Databáze: | OpenAIRE |
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