One of the primary barriers to the physical and structural characterization of membrane proteins is their often poor expression with standard methods. This project seeks to alleviate this bottleneck by creating E. coli strains that are more effective membrane protein producers. We plan to take a genetic approach, using a powerful selection for membrane protein expression that we have developed. By identifying and characterizing expression mutants we also plan learn more about the major barriers to expression. Our longer term goals are to apply the lessons learned here to other organisms, further broadening the range of membrane proteins that can be expressed at high levels.
The specific aims are:
Aim I. Find genes that (when over-expressed or mutated) can improve the production of poorly expressed membrane proteins and/or that redirect expression from inclusion bodies into membranes.
Aim II. Identify the genes/mutations.
Aim III. Employ targeted mutagenesis of the genes identified in Aim II to find even more effective expression mutants. Combine the most effective mutants to build a set of membrane protein expression strains.
Aim I V. Characterize the effects of the expression mutants on the steps of membrane protein biogenesis. Relevance to Public Health: Membrane proteins play significant roles in diseases ranging from cystic fibrosis to cancer, and are the targets of the vast majority of drugs. Our understanding of these diseases and our ability to develop new drugs has been hampered by our inability to obtain sufficient quantities of many membrane proteins for detailed study. This project is an effort to reduce this problem by converting bacteria into membrane protein production factories. ? ? ?
| Massey-Gendel, Elizabeth; Zhao, Anni; Boulting, Gabriella et al. (2009) Genetic selection system for improving recombinant membrane protein expression in E. coli. Protein Sci 18:372-83 |
| Blois, Tracy M; Bowie, James U (2009) G-protein-coupled receptor structures were not built in a day. Protein Sci 18:1335-42 |
