Maltose-Binding Protein as a Fusion Tag for the Localization and Purification of Cloned Proteins in Dictyostelium

✍ Scribed by Ralph Gräf

• Publisher: Elsevier Science
• Year: 2001
• Tongue: English
• Weight: 116 KB
• Volume: 289
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1006/abio.2000.4961

✦ Synopsis

encoding N-terminal-deleted regions of human progesterone receptor, in agreement with the observations that the cDNAs encoding progesterone receptor were detected by RT-PCR in the human ovary cDNA library, but not in the HeLa cell cDNA library. The ligandindependent colonies turned out to harbor cDNA encoding liver X receptor ␣ (LXR␣), liver X receptor ␤ (LXR␤), and retinoic acid receptor-like orphan receptor ␥ (ROR␥). No interactant other than nuclear receptors was obtained from this screening.

We showed that progesterone receptor was isolated from the human ovary cDNA library in response to ligand, while other nuclear receptors were also isolated, but in the absence of ligand. Moreover, by a preliminary experiment the cDNA clone encoding VDR was isolated from the human kidney library with low efficiency (K. Takeyama, unpublished results). It is therefore noteworthy that this screening system is valid also for isolating the cDNAs for heterodimeric nuclear receptors at least for VDR, LXRs, and RORtype receptors. However, as the interaction domain for SRC-1 is mapped to the C-terminal LBD, the cDNA clones in the libraries with high numbers beyond screening efficiency are absolutely required to encode at least the LBD of nuclear receptors. These findings indicate that the SRC-1 fragment serves as a specific interface for nuclear receptors, and hence this fragment serves as bait for screening nuclear receptors in this yeast two-hybrid system. By this method using the SRC-1 short fragment, one can trap nuclear receptors that are activated by a ligand of interest without aiming at any specified nuclear receptor. Therefore, we propose that this method offers another approach for evaluating the effect of drugs.