title: Enhancing the stability and solubility of the glucocorticoid receptor ligand-binding domain by high-throughput library screening.
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.
authors.affiliation: Institute of Biophysics and Physical Biochemistry, University of Regensburg, Universitätsstrasse 31, D-93053 Regensburg, Germany.
authors.forename: Tobias
authors.initials: T
authors.lastname: Seitz
authors.forename: Ralf
authors.initials: R
authors.lastname: Thoma
authors.forename: Guillaume A
authors.initials: GA
authors.lastname: Schoch
authors.forename: Martine
authors.initials: M
authors.lastname: Stihle
authors.forename: Jörg
authors.initials: J
authors.lastname: Benz
authors.forename: Brigitte
authors.initials: B
authors.lastname: D'Arcy
authors.forename: Andrea
authors.initials: A
authors.lastname: Wiget
authors.forename: Armin
authors.initials: A
authors.lastname: Ruf
authors.forename: Michael
authors.initials: M
authors.lastname: Hennig
authors.forename: Reinhard
authors.initials: R
authors.lastname: Sterner
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000235:genetics; D005434:Flow Cytometry; D015723:Gene Library; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics; D006801:Humans; D066298:In Vitro Techniques; D008024:Ligands; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011487:Protein Conformation; D015202:Protein Engineering; D055550:Protein Stability; D017434:Protein Structure, Tertiary; D011965:Receptors, Glucocorticoid / Q000737:chemistry* / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D012995:Solubility; D055672:Static Electricity; D053300:Tumor Necrosis Factor Ligand Superfamily Member 13 / Q000235:genetics
chemical_list: D008024:Ligands; D011965:Receptors, Glucocorticoid; D011993:Recombinant Fusion Proteins; D053300:Tumor Necrosis Factor Ligand Superfamily Member 13; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins