Journal of Korean Endocrine Society 1997;12(2):283-294.
Published online January 1, 2001.
Expression of Phospholipase C-B3 using Recombinant Baculovirus Expression System.
Do Joon Park
Abstract
BACKGROUND
Although phospholipase C (PLC)-B3 is thought to be a very important enzyme in intracellular signal transduction, the sophisticated and complicated purification steps make it difficult to obtain sufficient amount of protein to study regulation of its activity by G proteins or other proteins. In order to get large amount of PLC-B3 protein, I employed baculovirus expression system which is known to express large amount of functionally active proteins. METHODS: In order to make recombinant baculovirus which expresses PLC-B3 gene, partial cDNA of PLC-B3 which lacked 51 nucleotides was used to make full length PLC-B3 cDNA. By PCR, 5-end sequence of PLC-B3 was ligated into partial rat PLC-B3 cDNA and later cloned into pVL1393 transfer vector to make recombinant baculovirus. This recombinant baculovirus containing PLC-B3 sequence was used to infect Sf9 insect cells. RESULTS: Infection of Sf9 cells with recombinant baculovirus rendered expression of 152 kDa-PLC-B3 protein, which was confirmed by immunoblot assay and PLC activity assay. CONCLUSION: The whole length PLC-B3 cDNA was expressed in Sf9 cells using baculovirus expression system. By using it, homogeneous enzyme is expected to be purified to study precise activation and regulation mechanisms of PLC-B3.
Key Words: Phosphoinositide-specific phospholipase C (PI-PLC), Baculovirus, Signal transduction
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