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KMID : 0356620120270040282
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Journal of Korean Society of Endocrinology
2012 Volume.27 No. 4 p.282 ~ p.288
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Expression and Purification of Phospholipase C-β4, and Chimeric Phospholipase C and Characterization of Them
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Park Do-Joon
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Abstract
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Background: Phospholipase C-β4 (PLC-β4) is known to be one of the most important signal transducing molecules; however, its biophysical and chemical characteristics are not well known due to the difficulty in purifying PLC-β4 from bovine retina. In the present study, we used the baculovirus expression system in order to express and purify large amounts of PLC-β4. With this system, we also tried to produce chimeric PLC-β3/β4 and PLC-β4/β3 protein in order to study the structure-activity relationship between N terminal and C terminal portion of PLC-βs.
Methods: I cloned PLC-β4 to the baculovirus expression system by the polymerase chain reaction method and infected the PLC-β4 to Sf9 cells. I purified recombinant PLC-β4 proteins using sequential high performnance liquid chromatography (HPLC) by using the TSK phenyl-5PW column and the TSK heparin-5PW column. With this similar method, I was able to express chimeric PLC-β3/β4 and PLC-β4/β3 proteins.
Results: With the two step HPLC, I was able to purify PLC-β4 by 30-fold; this purified PLC-β4 contained PLC activity. I also expressed chimeric PLC-β3/β4 and PLC-β4/β3 using the baculovirus system, and their expression was confirmed by the immunoblot method. However, chimeric PLC-β4/β3 did not show PLC activity, while chimeric PLC-β3/β4 retained its PLC-activity.
Conclusion: Expression of chimeric PLC-β4 using the baculovirus system was an efficient method to obtain a large amount of protein. Moreover, this expression and purification method would be useful in studying the physical and chemical characteristics of this protein. In my study using chimeric PLC-β protein by swapping the N terminal and C terminal portions of PLC-β3 and β4, chimeric protein lost its activity completely in PLC-β4/β3 chimera. This result suggested a minute change in the tertiary structure of the protein, which may significantly affect its function.
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KEYWORD
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Baculovirus, Chimera, Isolation and purification, Phospholipase C beta
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