Seitz, Tobias, Thoma, Ralf, Schoch, Guillaume A., Stihle, Martine, Benz, Jörg, D'Arcy, Brigitte, Wiget, Andrea, Ruf, Armin, 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.
Full text not available from this repository.
at PubMed
at publisher (via DOI)
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 ...
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 °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.
Export bibliographical data
| Item type: | Article |
|---|
| Date: | 2010 |
|---|
| Institutions: | Biology, Preclinical Medicine > Institut für Biophysik und physikalische Biochemie > Prof. Dr. Reinhard Sterner |
|---|
| Identification Number: | | Value | Type |
|---|
| 20850457 | PubMed ID | | 10.1016/j.jmb.2010.08.048 | DOI |
|
|---|
| Classification: | | Notation | Type |
|---|
| Amino Acid Sequence | MESH | | Amino Acid Substitution | MESH | | Animals | MESH | | Base Sequence | MESH | | Binding Sites/genetics | MESH | | Crystallography, X-Ray | MESH | | Escherichia coli/genetics | MESH | | Flow Cytometry | MESH | | Gene Library | MESH | | Green Fluorescent Proteins/genetics | MESH | | Humans | MESH | | Ligands | MESH | | Mice | MESH | | Models, Molecular | MESH | | Molecular Sequence Data | MESH | | Mutagenesis | MESH | | Protein Conformation | MESH | | Protein Engineering | MESH | | Protein Stability | MESH | | Protein Structure, Tertiary | MESH | | Receptors, Glucocorticoid/genetics | MESH | | Recombinant Fusion Proteins/genetics | MESH | | Sequence Homology, Amino Acid | MESH | | Solubility | MESH | | Static Electricity | MESH | | Tumor Necrosis Factor Ligand Superfamily Member 13/genetics | MESH |
|
|---|
| Dewey Decimal Classification: | 500 Science > 570 Life sciences |
|---|
| Status: | Published |
|---|
| Refereed: | Yes, this version has been refereed |
|---|
| Created at the University of Regensburg: | Yes |
|---|
| Item ID: | 20721 |
|---|
Altmetric