High level expression in Escherichia coli of the DNA-binding domain of the glucocorticoid receptor in a functional form utilizing domain-specific cleavage of a fusion protein.
A fragment comprising the DNA-binding domain of the human glucocorticoid receptor has been expressed in a functional form in Escherichia coli as a fusion protein with protein A from Staphylococcus aureus. The DNA-binding domain was purified to apparent homogeneity by affinity chromatography on IgG-Sepharose and DNA-cellulose, a purification scheme which does not involve denaturation of the protein at any step. The DNA-binding domain was separated from the protein A part of the fusion protein by domain-specific enzymatic cleavage with chymotrypsin while immobilized on IgG-Sepharose. The recombinant protein has been characterized by amino acid analysis, NH2- and COOH-terminal sequence analysis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and reactivity to iodoacetate and was found to correspond to the primary structure derived from the cDNA sequence. DNase I footprinting showed that the purified recombinant protein bound to the same DNA sequences on the mouse mammary tumor virus long terminal repeat as glucocorticoid receptor purified from rat liver does. About 10 times more recombinant protein, on a molar basis, was needed to obtain the same level of protection. However, the protection of the three different footprints (1.3, 1.4, and 1.5') by the recombinant protein differed greatly from that of the natural receptor, with virtually no protection of footprint 1.4. This indicates cooperative binding of the natural receptor to adjacent footprints, dependent on other regions of the receptor than the DNA-binding domain.
Dahlman
K
K
Department of Medical Nutrition, Karolinska Institute, Huddinge University Hospital, Sweden.
Strömstedt
P E
PE
Rae
C
C
Jörnvall
H
H
Flock
J I
JI
Carlstedt-Duke
J
J
Gustafsson
J A
JA
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D011965:Receptors, Glucocorticoid / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism
D004268:DNA-Binding Proteins; D011965:Receptors, Glucocorticoid; D011993:Recombinant Fusion Proteins; D004247:DNA