We recently developed a display method for the directed evolution of integral membrane proteins in the inner membrane of Escherichia coli for higher expression and stability. For the neurotensin receptor 1, a G-protein-coupled receptor (GPCR), we had evolved a mutant with a 10-fold increase in functional expression that largely retains wild-type binding and signaling properties and shows higher stability in detergent-solubilized form. We have now evolved three additional human GPCRs. Unmodified wild-type receptor cDNA was subjected to successive cycles of mutagenesis and fluorescence-activated cell sorting, and functional expression could be increased for all three GPCR targets. We also present a new stability screening method in a 96-well assay format to quickly identify evolved receptors showing increased thermal stability in detergent-solubilized form and rapidly evaluate them quantitatively. Combining the two methods turned out to be very powerful; even for the most challenging GPCR target-the tachykinin receptor NK1, which is hardly expressed in E. coli and cannot be functionally solubilized-receptor mutants that are functionally expressed at 1 mg/l levels in E. coli and are stable in detergent solution could be quickly evolved. The improvements result from cumulative small changes in the receptor sequence. This combinatorial approach does not require preconceived notions for designing mutations. Our results suggest that this method is generally applicable to GPCRs. Existing roadblocks in structural and biophysical studies can now be removed by providing sufficient quantities of correctly folded and stable receptor protein.
|Original language||English (US)|
|Number of pages||17|
|Journal||Journal of Molecular Biology|
|State||Published - May 13 2011|
Bibliographical noteFunding Information:
We acknowledge the use of and the very professional help by the staff of the Flow Cytometry Laboratory of the Institute for Biomedical Engineering, University of Zurich and Eidgenössische Technische Hochschule Zurich. This work was supported by a predoctoral fellowship to I.D. from the Forschungskredit of the University of Zurich and by the Swiss National Science Foundation (National Center of Competence in Research in Structural Biology) to A.P.
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- directed evolution
- expression in E. coli
- protein engineering