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Published on: November 8, 2019
Expression and purification of OsVDAC4
Ashwini Godbole1, Ashvini K Dubey2, Rohan Mitra1
1National Centre for Biological Sciences, TIFR, Bangalore, India.
Abstract:
The voltage-dependent anion channel (VDAC), a major component of the mitochondrial outer membrane, has emerged as an important player in cell function, survival, and death signaling. VDAC function is modulated by its interaction with proteins such as hexokinase, adenine nucleotide translocator, and apoptotic proteins like Bax. Monitoring the activity of VDAC and its modulation in the complex cellular milieu is fraught with complications. Minimizing the number of components in the study is one approach to teasing apart various aspects of its function. In this chapter, we have described detailed protocols for the purification of a rice VDAC isoform, OsVDAC4 after overexpression in a bacterial system. The protein is solubilized with LDAO and then reconstituted into liposomes or planar bilayers to verify its competence to fold into a functionally active form.
Insights
This study details the purification of rice voltage-dependent anion channel 4 (OsVDAC4) from a bacterial system. The purified protein was reconstituted into liposomes and planar bilayers to confirm its functional activity.
Area of Science:
- Mitochondrial biology
- Ion channel research
- Plant biochemistry
Background:
- The voltage-dependent anion channel (VDAC) is crucial in mitochondrial outer membrane functions, including cell survival and death signaling.
- VDAC activity is modulated by interactions with various cellular proteins, complicating direct study.
- Investigating VDAC function often requires simplified systems to isolate specific roles.
Purpose of the Study:
- To establish a protocol for purifying a specific rice VDAC isoform, OsVDAC4.
- To demonstrate a method for assessing the functional activity of purified OsVDAC4.
Main Methods:
- Overexpression of rice OsVDAC4 in a bacterial system.
- Solubilization of OsVDAC4 using LDAO detergent.
- Reconstitution of purified OsVDAC4 into liposomes and planar lipid bilayers.
Main Results:
- Successful purification of recombinant OsVDAC4 was achieved.
- Reconstituted OsVDAC4 exhibited functional activity in liposome and planar bilayer assays.
- The protocol allows for the study of OsVDAC4 in a controlled, reconstituted system.
Conclusions:
- The described method enables the purification and functional assessment of rice OsVDAC4.
- This approach simplifies the study of VDAC function by minimizing confounding cellular components.
- The findings provide a foundation for further investigations into OsVDAC4's role in plant cell physiology.
