PCR expression mutagenesis: a high-throughput mutation assay applied to the glucocorticoid receptor ligand-binding domain.
Glucocorticoid receptors (GRs) are extensively studied members of the steroid hormone receptor superfamily that regulate the transcription rates of numerous genes. Notwithstanding, the role of each GR amino acid in the various steps of transactivation is still unknown. A recent report shows that linear DNA has the same capacity as super-helical plasmid DNA for gene expression in transient transfection assays. Based on this observation, we describe a high-throughput assay to analyze a large set of alanine point mutations that are introduced by two rounds of PCR. The PCR products are then directly transfected into cells. This PCR expression mutagenesis (PEM) technique is used to identify several new residues of the GR ligand binding domain that influence ligand binding and/or transactivation. PEM thus provides a quick method for screening large quantities of mutant proteins. In combination with automation, PEM provides a more rapid and efficient tool for probing the role of each amino acid in the biological functions of a given protein.
Chen
Jun
J
Steroid Hormones Section, NIDDK/LMCB, National Institutes of Health, Bethesda, MD, USA. jchen@usuhs.mil
Blackford
John A
JA
Simons
S Stoney
SS
D000818:Animals; D001665:Binding Sites / Q000235:genetics; D019556:COS Cells; D002460:Cell Line; D008024:Ligands; D008958:Models, Molecular; D016296:Mutagenesis; D017354:Point Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*; D017434:Protein Structure, Tertiary; D011965:Receptors, Glucocorticoid / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015533:Transcriptional Activation; D014162:Transfection
D008024:Ligands; D011965:Receptors, Glucocorticoid; D011994:Recombinant Proteins