Expression and Purification of Phospholipase C-beta4, and Chimeric Phospholipase C and Characterization of Them.
10.3803/EnM.2012.27.4.282
- Author:
Do Joon PARK
1
Author Information
1. Department of Internal Medicine, Seoul National University College of Medicine, Seoul, Korea. djpark@snu.ac.kr
- Publication Type:Original Article
- Keywords:
Baculovirus;
Chimera;
Isolation and purification;
Phospholipase C beta
- MeSH:
Baculoviridae;
Chimera;
Chromatography, High Pressure Liquid;
Chromatography, Liquid;
Clone Cells;
Phospholipase C beta;
Phospholipases;
Polymerase Chain Reaction;
Proteins;
Retina;
Sf9 Cells;
Structure-Activity Relationship;
Type C Phospholipases
- From:Endocrinology and Metabolism
2012;27(4):282-288
- CountryRepublic of Korea
- Language:Korean
-
Abstract:
BACKGROUND: Phospholipase C-beta4 (PLC-beta4) 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-beta4 from bovine retina. In the present study, we used the baculovirus expression system in order to express and purify large amounts of PLC-beta4. With this system, we also tried to produce chimeric PLC-beta3/beta4 and PLC-beta4/beta3 protein in order to study the structure-activity relationship between N terminal and C terminal portion of PLC-betas. METHODS: I cloned PLC-beta4 to the baculovirus expression system by the polymerase chain reaction method and infected the PLC-beta4 to Sf9 cells. I purified recombinant PLC-beta4 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-beta3/beta4 and PLC-beta4/beta3 proteins. RESULTS: With the two step HPLC, I was able to purify PLC-beta4 by 30-fold; this purified PLC-beta4 contained PLC activity. I also expressed chimeric PLC-beta3/beta4 and PLC-beta4/beta3 using the baculovirus system, and their expression was confirmed by the immunoblot method. However, chimeric PLC-beta4/beta3 did not show PLC activity, while chimeric PLC-beta3/beta4 retained its PLC-activity. CONCLUSION: Expression of chimeric PLC-beta4 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-beta protein by swapping the N terminal and C terminal portions of PLC-beta3 and beta4, chimeric protein lost its activity completely in PLC-beta4/beta3 chimera. This result suggested a minute change in the tertiary structure of the protein, which may significantly affect its function.
