Membrane proteins represent large fractions of the proteomes of all organisms and are responsible for diverse functions in cells. The ability to study these proteins from a structural and biochemical standpoint is often contingent upon overexpression of the protein in a host such as E. coli. Using strains that either deplete or overproduce molecules responsible for membrane biogenesis in E. coli (the proteins Ffh, FtsY, and YidC and the 4.5S RNA), we will systematically evaluate the effects of these molecules on the expression of a polytopic E. coli membrane protein YeaS. The analysis will be carried out using a novel flow cytometric assay, allowing for high-throughput quantitative determination of expression level. The same assay will be used in a genetic screen to identify novel factors influencing membrane protein biogenesis. A unified experimental and bioinformatic approach will also used to study sequence elements in membrane proteins that affect expression level. Lastly, the expression of human cannabinoid receptor 1 (CB1), a membrane-bound G protein-coupled receptor (GPCR) found in the central nervous system of profound interest to the pharmaceutical industry, will be optimized for expression in the strains of E. coli described above. In so doing, a robust platform for the high-level heterologous expression of membrane proteins in E. coli will be developed. Such a platform will be of great utility to researchers interested in the structural and biochemical properties of membrane proteins. ? ? ?
| Link, A James; Jeong, Ki Jun; Georgiou, George (2007) Beyond toothpicks: new methods for isolating mutant bacteria. Nat Rev Microbiol 5:680-8 |
