Endocrinol Metab.  2012 Dec;27(4):282-288. 10.3803/EnM.2012.27.4.282.

Expression and Purification of Phospholipase C-beta4, and Chimeric Phospholipase C and Characterization of Them

• Park DJ

• KMID: 1973790
• DOI: http://doi.org/10.3803/EnM.2012.27.4.282

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.

Keyword

Baculovirus; Chimera; Isolation and purification; Phospholipase C beta

MeSH Terms

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
Phospholipase C beta
Phospholipases
Proteins
Type C Phospholipases

Figure

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  Fig. 1 Sequences of chimeric phospholipase C (PLC)-β3/β4 and PLC-β4/β3. Each alphabet letter represents single amino acid. Underlined letters mean swapped amino acids between PLC-β3 and PLC-β4.

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  Fig. 2 Purification of phospholipase C (PLC)-β4 on TSK phenyl-5PW column. Solid line shows the absorbance at 280 nm which represents the concentration of the eluted protein. Open circles represent the activity of PLC-β3 as cpm, and the dotted line shows the KCl gradient.

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  Fig. 3 Purification of phospholipase C (PLC)-β4 on TSK heparin-5PW column. Solid line shows the absorbance at 280 nm which represents the concentration of the eluted protein. Open circles represent the activity of PLC-β3 as cpm, and the dotted line shows the NaCl gradient.

• 

  Fig. 4 Immunoblot of chimeric phospholipase C (PLC)-β3/β4 and PLC-β4/β3 with antibodies against C terminal of PLC-β3, β4, N terminal of PLC-β3 or β4. Sf9 cells infected with recombinant chimeric PLC-β3/β4 or PLC-β4/β3 were disrupted with sonicator, and 20 µg protein of each homogenate was separated on 6% sodium dodecyl sulfate-polyacrylamide gel. (A) Panel A was probed with antibodies against N terminal PLC-β3. (B) Panel B was probed with antibodies against C terminal PLC-β3. (C) Panel C was probed with antibodies against N terminal PLC-β4. (D) Panel D was probed with antibodies against C terminal PLC-β4. The molecular weight of chimeric PLC-β3/β4 is lower than chimeric PLC-β4/β3, and its cognate bands were revealed by antibodies against N terminal PLC-β3 and antibodies against C terminal PLC-β4 (A, D). Chimeric PLC-β4/β3 bands were revealed by antibodies against C terminal PLC-β3 and antibodies against N terminal PLC-β4 (B, C).

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