期刊
PROTEIN SCIENCE
卷 21, 期 2, 页码 279-288出版社
WILEY
DOI: 10.1002/pro.2014
关键词
ATP synthase; protein NMR; cell-free synthesis; membrane protein
资金
- Natural Sciences and Engineering Research Council of Canada Discovery
- United States Public Health Service [GM23105]
- NIH
- The University of Wisconsin
- National Science Foundation
- Department of Agriculture
NMR structure determination of large membrane proteins is hampered by broad spectral lines, overlap, and ambiguity of signal assignment. Chemical shift and NOE assignment can be facilitated by amino acid selective isotope labeling in cell-free protein synthesis system. However, many biological detergents are incompatible with the cell-free synthesis, and membrane proteins often have to be synthesized in an insoluble form. We report cell-free synthesis of subunits a and c of the proton channel of Escherichia coli ATP synthase in a soluble form in a mixture of phosphatidylcholine derivatives. In comparison, subunit a was purified from the cell-free system and from the bacterial cell membranes. NMR spectra of both preparations were similar, indicating that our procedure for cell-free synthesis produces protein structurally similar to that prepared from the cell membranes.
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