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PCR expression mutagenesis: a high-throughput mutation assay applied to the glucocorticoid receptor ligand-binding domain.

Abstract：

: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.

journal_name

Biochem Biophys Res Commun

journal_title

Biochemical and biophysical research communications

authors

Chen J,Blackford JA Jr,Simons SS Jr

doi

10.1016/j.bbrc.2004.07.047

subject

Has Abstract

pub_date

2004-09-03 00:00:00

pages

893-9

issue

4

eissn

0006-291X

issn

1090-2104

pii

S0006-291X(04)01506-2

journal_volume

321

pub_type

杂志文章

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