| Research Abstract |
Transcription factors or membrane receptor proteins (e. g., the G protein-coupled receptor [GPCR]) are considered to precisely regulate physiological processes by forming complexes with a number of proteins. As the amounts of these proteins, which are important for cellular responses to stimuli, are very small, and purification is difficult, a dynamic analysis of their functions in vivo has not yet been undertaken. In this study, we have developed a method for the functional analysis of membrane proteins expressed and reconstituted on the baculoviruses (Sakihama T., J Biotechnol, 2008). Furthermore, we have developed a method to analyze the dynamism of protein-protein interactions for the endogenous proteins, by fixing the high affinity monoclonal antibodies obtained using the protein-expressing baculovirus (Saitoh R., et. al., JIM, 2007) to low noise magnetic beads, and performing a mass spectrometric analysis.
In other veins, a novel regulation pathway mediated by angiotensin was foun
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d by utilizing monoclonal antibodies of angiotensin type II receptor (AT2) and angiotensin receptor-interacting proteins (ATIPs), prepared using baculovirus. We also found that the WTAP (WT1 associated protein) is involved in the stability of cyclin A2 mRNA thereby regulates the cell cycle. We have reported in the meetings that it is possible to detect endogenous HNF4alpha, a nuclear receptor which is important for liver and pancreas formation, from one 10 cm dish cell sample, and to identify over one hundred associating proteins for HNF4alpha, by establishing a method for affinity purification mass spectrometry using magnetic beads and high-affinity antibodies. The sensitive identification of very small amounts of endogenous proteins makes it possible to analyze the physiological regulation dynamics. The method described above will be a powerful tool for protein modification or protein-protein interaction assay for proteins involved in the pathology of various diseases and pharmacologic actions. Less
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