BIOT 142 |
| Membrane proteins present unparalleled challenges for structural biology initiatives and nascent functional genomics efforts. To overcome some of the current hurdles in obtaining sufficient quantities of membrane proteins for these structural and functional studies, we are in the process of employing and optimizing a novel host/vector system based upon the Rhodobacter species of photosynthetic bacteria. This system exploits the unique physiology of this prokaryotic host whereby strongly-induced heterologous expression of the hydrophobic target proteins can be coordinated with induction of newly developing membranes, thereby favoring membrane insertion of natively folded polypeptides rather than inclusion body formation. In Rhodobacter, coordinated synthesis of new membrane and target membrane protein is induced by oxygen and/or light. In the former case, autoinduction by oxygen occurs as the cell density of the culture increases. Maximal expression is achieved with the promoter that responds to both light and oxygen. A series of vectors based upon these promoter types has been constructed and modifications to them include those that allow for variety in composition and placement of affinity tags, ligation-independent cloning, and affinity tag cleavage. Several host strains are available that produce induced membranes that carry the full complement of native host membrane proteins or carry deletions of one or more of those native transmembrane complexes. The variability in the hosts explores the potential for increased production in “partially empty” membrane and also facilitates various growth modes. Preliminary data suggest that many target membrane proteins from a variety of organisms can be expressed and many can be produced and purified at levels that equal or exceed those of native membrane protein complexes (10 mg/L). Standardized strategies for the semi-automated cloning, semi-automated purification, and heavy-atom derivatization of affinity-tagged, target membrane proteins will also be presented. |
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Biophysical and Biomolecular Symposium: Challenges to Membrane Protein Production and Characterization
8:00 AM-10:55 AM, Tuesday, 12 September 2006 Hilton San Francisco -- Imperial B Ballroom, Oral
Division of Biochemical Technology |