Seitz, Tobias and Thoma, Ralf and Schoch, Guillaume A. and Stihle, Martine and Benz, Joerg and D'Arcy, Brigitte and Wiget, Andrea and Ruf, Armin and Hennig, Michael and Sterner, Reinhard (2010) Enhancing the Stability and Solubility of the Glucocorticoid Receptor Ligand-Binding Domain by High-Throughput Library Screening. JOURNAL OF MOLECULAR BIOLOGY, 403 (4). pp. 562-577. ISSN 0022-2836, 1089-8638
Full text not available from this repository. (Request a copy)Abstract
The human glucocorticoid receptor ligand-binding domain (hGR-LBD) is an important drug target for the treatment of various diseases. However, the low intrinsic stability and solubility of hGR-LBD have rendered its purification and biophysical characterization difficult. In order to overcome these problems, we have stabilized hGR-LBD by a combination of random mutagenesis and high-throughput screening using fluorescence-activated cell sorting (FACS) with enhanced green fluorescent protein (eGFP) as folding reporter. Two plasmid-encoded gene libraries of hGR-LBD fused to the egfp gene were expressed in Escherichia coli, followed by eight rounds of FACS screening, in each of which 10(8) cells were analyzed. The hgr-lbd mutants isolated by this approach contained numerous amino acid exchanges, and four beneficial ones (A605V, V702A, E705G, and M752T) were followed up in detail. Their characterization showed that the fluorescence of hGR-LBD-eGFP fusions is correlated linearly with the stability and solubility of hGR-LBD in the absence of eGFP. When combined, the four exchanges increased the thermal stability of hGR-LBD by more than 8 degrees C and enhanced its purification yield after expression in E. coli by about 26-fold. The introduction of three beneficial exchanges into the homologous ligand-binding domain of mouse enabled its X-ray structure determination at high resolution, which showed how the exchanges stabilize the protein and revealed atomic details that will guide future drug design. Our results demonstrate that large eGFP fusion libraries can be screened by FACS with extreme sensitivity and efficiency, yielding stabilized eukaryotic proteins suitable for biophysical characterization and structure determination. (C) 2010 Elsevier Ltd. All rights reserved.
| Item Type: | Article |
|---|---|
| Uncontrolled Keywords: | GREEN FLUORESCENT PROTEIN; DIRECTED ENZYME EVOLUTION; RAY CRYSTAL-STRUCTURE; HETEROLOGOUS EXPRESSION; MUTAGENESIS; THERMOSTABILITY; MECHANISMS; ANTAGONISM; SUFFICIENT; SOFTWARE; FACS; GFP; folding reporter; library screening; glucocorticoid receptor |
| Subjects: | 500 Science > 570 Life sciences |
| Divisions: | Biology, Preclinical Medicine > Institut für Biophysik und physikalische Biochemie > Prof. Dr. Reinhard Sterner |
| Depositing User: | Dr. Gernot Deinzer |
| Date Deposited: | 07 Jul 2020 05:45 |
| Last Modified: | 07 Jul 2020 05:45 |
| URI: | https://pred.uni-regensburg.de/id/eprint/23889 |
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