Factors That EnhanceEscherichia coli-Expressed TRβ Binding to T₃and DNA

Abstract

Thyroid hormone receptors (TRs) recently have been produced in E. coli by several laboratories. We produced E. coli-expressed human TR beta using the histidine/fusion protein system. Surprisingly, we observed that reticulocyte lysate, nonspecific proteins, and 1% Triton X dramatically increased both the T3- and DNA-binding activities of human TR beta. These studies demonstrate that there are a number of factors that will enhance ligand and DNA binding of E. coli-expressed TR beta. Addition of these factors to reaction samples containing E. coli-expressed TRs will help to optimize measurement conditions. These findings also suggest that experiments in which cellular proteins are added to highly purified TR preparations may require controls to eliminate contributions by nonspecific proteins.

Effect of mutations in the β1‐thyroid hormone receptor on the inhibition of T3 binding by desethylamiodarone

“…was added the cells were grown for another 1.5 h. Cells were then lysed by sonification in PBS (2×20 s, 50 W on ice). Each receptor protein preparation was purified using glutathione‐Sepharose 4B affinity resin (Pharmacia Biotech, Sweden) according to the manufacturer's instructions with the following modifications [9–11]: Triton X‐100 was used in a final concentration of 0.5%, PBS used as a wash solution contained 2 mM DTT and 0.2 mM PMSF. The proteins were stored at a high concentration in incubation buffer (20 mM Tris‐HCl, 0.25 M sucrose, 1 mM EDTA, 50 mM NaCl, 5% (v/v) glycerol, 5 mM DTT pH 7.6) in liquid nitrogen, thawed on ice and diluted to the desired concentration just before use.…”