A homogeneous in vitro functional assay for estrogen receptors: coactivator recruitment.

Estrogen receptor (ER)-mediated gene transcription occurs via the formation of a multimeric complex including ligand-activated receptors and nuclear coactivators. We have developed a homogeneous in vitro functional assay to help study the ligand-dependent interaction of ERs with various nuclear coactivators. The assay consists of FLAG-tagged ERalpha or ERbeta ligand binding domain (LBD), a biotinylated coactivator peptide, europium-labeled anti-FLAG antibody, and streptavidin-conjugated allophycocyanin. Upon agonist binding, the biotinylated coactivator peptide is recruited to FLAG-tagged ER LBD to form a complex and thus allow fluorescence resonance energy transfer (FRET) to occur between europium and allophycocyanin. Compounds with estrogen antagonism block the agonist-mediated recruitment of a coactivator and prevent FRET. The assay was used to evaluate the preference of ERs for various coactivators and ligands. Both ERalpha and ERbeta exhibited strong preferences for coactivator peptides corresponding to steroid receptor coactivator-1 and PPARgamma coactivor-1 vs. peroxisome proliferator-activated receptor-interacting protein and cAMP response element binding protein-binding protein. 17beta-Estradiol acted as a nonselective agonist for ERalpha and ERbeta. Genistein showed full agonism for ERalpha and only partial agonism for ERbeta, but with higher potency for ERbeta than ERalpha. Both raloxifene and tamoxifen behaved as full antagonists in this functional assay. The results obtained using compounds with a wide range of potency correlated well with those from a cell-based reporter gene assay. Therefore, this simple in vitro functional assay is predictive of ligand-dependent transactivation function of the receptor and, as such, is useful in nuclear receptor applications including mechanistic studies.

Improvements to the throughput of recombinant protein expression in the baculovirus/insect cell system.

Recombinant baculoviruses have proved to be a very useful means to express many proteins over the last 20 years. Since their introduction, there have been a number of significant improvements that have simplified and speeded up the construction of baculoviruses. One of the most commonly used methods relies upon recombination with the baculovirus genome maintained in Escherichia coli. In this paper, we report the conversion of nearly all the steps in this process including the expression testing and purification to a multi-well plate format. This enables a significant increase in the number of constructs that can be processed in a shorter period of time and an order of magnitude increase in the number of expression conditions that can be analysed. A key step in our process is that the transfection is done in suspension rather than adherent cells, which gives a much higher virus titre than in the standard methods.