Purification of human c-erb A beta protein.
Human thyroid hormone receptor (c-erb A protein) produced by Escherichia coli expression vector plasmid was purified sequentially using polyethylenimine precipitation of DNA, hydroxylapatite column chromatography, ammonium sulphate precipitation, Sephacryl S-300 gel filtration and mono Q-Sepharose column chromatography. These column procedures resulted in 41.3-fold purification of 3,5,3'-tri-iodo-L-thyronine (T3) binding activity over the initial E. coli extract. Purified protein as well as crude preparation showed high-affinity binding to T3. The c-erb A protein enriched by column purification was further purified by electroelution after electrophoresis. Rabbit antibody against the c-erb A protein was prepared and used for the Western blotting analysis. The antibody recognized c-erb A protein but not the bacterial proteins in crude E. coli extract. When partially purified rat hepatic nuclear thyroid hormone receptor was analysed, a 56 kDa receptor was specifically recognized by the antibody.
Ichikawa
K
K
Department of Gerontology, Endocrinology and Metabolism, Shinshu University School of Medicine, Matsumoto, Japan.
Hashizume
K
K
Nishii
Y
Y
Takeda
T
T
Kobayashi
M
M
Suzuki
S
S
Yamada
T
T
D000906:Antibodies / Q000276:immunology; D001665:Binding Sites; D015153:Blotting, Western; D002845:Chromatography; D002850:Chromatography, Gel; D004247:DNA / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008099:Liver / Q000737:chemistry; D010957:Plasmids; D011518:Proto-Oncogene Proteins / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D011988:Receptors, Thyroid Hormone / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D011994:Recombinant Proteins / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D014285:Triiodothyronine, Reverse / Q000378:metabolism
D000906:Antibodies; D011518:Proto-Oncogene Proteins; D011988:Receptors, Thyroid Hormone; D011994:Recombinant Proteins; D014285:Triiodothyronine, Reverse; D004247:DNA