High yield cell-free production of integral membrane proteins without refolding or detergents
| Autor: | Jessica J. Wuu, James R. Swartz |
|---|---|
| Rok vydání: | 2007 |
| Předmět: |
Protein Folding
Detergents Biophysics Transporter Biochemistry Antiporters Integral membrane protein 03 medical and health sciences Bacterial Proteins 030304 developmental biology DNA Primers 0303 health sciences Cell-free protein synthesis In vitro protein synthesis biology Base Sequence Cell-Free System 030302 biochemistry & molecular biology Peripheral membrane protein Membrane Proteins Cell Biology Membrane transport Transmembrane protein Transport protein Membrane protein Chaperone (protein) biology.protein Electrophoresis Polyacrylamide Gel |
| Zdroj: | Biochimica et biophysica acta. 1778(5) |
| ISSN: | 0006-3002 |
| Popis: | Integral membrane proteins act as critical cellular components and are important drug targets. However, difficulties in producing membrane proteins have hampered investigations of structure and function. In vivo production systems are often limited by cell toxicity, and previous in vitro approaches have required unnatural folding pathways using detergents or lipid solutions. To overcome these limitations, we present an improved cell-free expression system which produces high yields of integral membrane proteins without the use of detergents or refolding steps. Our cell-free reaction activates an Escherichia coli-derived cell extract for transcription and translation. Purified E. coli inner membrane vesicles supply membrane-bound components and the lipid environment required for insertion and folding. Using this system, we demonstrated successful synthesis of two complex integral membrane transporters, the tetracycline pump (TetA) and mannitol permease (MtlA), in yields of 570+/-50 microg/mL and 130+/-30 microg/mL of vesicle-associated protein, respectively. These yields are up to 400 times typical in vivo concentrations. Insertion and folding of these proteins are verified by sucrose flotation, protease digestion, and activity assays. Whereas TetA incorporates efficiently into vesicle membranes with over two-thirds of the synthesized protein being inserted, MtlA yields appear to be limited by insufficient concentrations of a membrane-associated chaperone. |
| Databáze: | OpenAIRE |
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