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Effects of Zinc and Other Divalent Metals on Deoxyribonucleic Acid Binding and Hormone-Binding Activity of Human al Thyroid Hormone Receptor Expressed in Escherichia coli^*

Thyroid Study Unit, Department of Medicine, University of Chicago Chicago, Illinois 60637

Address all correspondence and requests for reprints to: Leslie J.DeGroot, M.D., Thyroid Study Unit, Box 138, University of Chicago,5841 South Maryland Avenue, Chicago, Illinois 60637.

Abstract

Full-length human thyroid hormone receptor al (hTRl) was expressed in Escherichia coli using a T7 expression system. While present in large amounts, the receptor was highly enriched in the insoluble fraction after cell lysis. We describe here the successful solubilization and refolding of the expressed receptor in a functional form in the presence of Zn²⁺. Using a DNA-cellulose binding assay and gel shift assay, we found that treatment of expressed receptor with 1 mM EDTA in the denaturing agent (5 M guanidine-HCl) results in the formation of aporeceptor that does not specifically recognize target DNA, while it does retain T₃-binding activity. This aporeceptor recovered DNA-binding activity by adding Zn²⁺ during refolding. Zinc-induced restoration of DNA-binding activity occurred in a dose- and time-dependent manner. Moreover, once recovered, this DNA-binding activity persisted without Zn²⁺, even in the presence of 1 mM EDTA. These data indicate that the hTRal molecule has a high affinity for Zn²⁺, and this metal coordination is essential for proper folding of TR protein into its native active structure. (Endocrinology 129: 3027–3033,1991)

Footnotes

^* This work was supported by USPHS Grants DK-13377 and DK-27384, March of Dimes Birth Defects Foundation Grant 1-1166, the Boots Pharmaceutical Co., and the David Wiener Research Fund.

Received June 4, 1991.

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