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Korean Society for Biochemistry and Molecular Biology (생화학분자생물학회)
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Expression of Enzymatically-active Phospholipase Cγ2 in E.coli

  • Ozdener, Fatih (Department of Pharmacology, Temple University Medical School) ;
  • Kunapuli, Satya P. (Sol Sherry Thrombosis Research Center, Temple University Medical School) ;
  • Daniel, James L. (Sol Sherry Thrombosis Research Center, Temple University Medical School)
  • Published : 2002.09.30
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Abstract

Phospholipase C-gamma-2 ($PLC{\gamma}2$) activation is a key signaling event for many cell functions. In order to delineate the pathways that lead to $PLC{\gamma}2$ activation, we devised a quick method for obtaining sufficient $PLC{\gamma}2$. We obtained the full-length cDNA for human $PLC{\gamma}2$ 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-$PLC{\gamma}2$ 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 $PLC{\gamma}2$ were found to catalyze the hydrolysis of phosphatidylinositol 4,5 bisphosphate. Similar to previous reports on $PLC{\gamma}2$ that is isolated from mammalian tissue, the recombinant enzyme was $Ca^{2+}$ dependent with optimal activity at 1-10 uM $Ca^{2+}$.

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References

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