Expression of Enzymatically - active Phospholipase Cγ2 in E. coli = Expression of Enzymatically - active Phospholipase Cγ2 in E. coli

다국어 초록 (Multilingual Abstract)

Phospholipase C-gamma-2 (PLCγ2) activation is a key signaling event for many cell functions. In order to delineate the pathways that lead to PLCγ2 activation, we devised a quick method for obtaining sufficient PLCγ2. We obtained the full-length cDNA for human PLCγ2 and expressed it in E. coli using the expression vector pT5T. To enhance the protein expression, tandem AGG-AGG arginine codons at the amino acid positions 1204-1205 were replaced by CGG-CGG arginine codons. The protein expression was detected in a Western blot analysis by both anti-PLCγ2 antibodies and the antibodies that are raised against the tripeptide epitope (Glu-Glu-Phe) tag that are genetically-engineered to its carboxyl terminal. Crude lysates that were prepared from bacteria that express PLCγ2 were found to catalyze the hydrolysis of phosphatidylinositol 4,5 bisphosphate. Similar to previous reports on PLCγ2 that is isolated from mammalian tissue, the recombinant enzyme was Ca^2+ dependent with optimal activity at 1-10 uM Ca^2+.