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Physiological functions of hBAX

Bax is a pro-apoptotic Bcl-2 protein. It promotes apoptosis by competing with Bcl-2 proper.

Upon initiation of apoptotic signaling, Bax undergoes a conformation shift, and inserts into organelle membranes, primarily the outer mitochondrial membrane.Bax is believed to interact with, and induce the opening of the mitochondrial voltage-dependent anion channel, VDAC. Alternatively, growing evidence suggest that activated Bax and/or Bak form an oligomeric pore, MAC in the outer membrane. This results in the release of cytochrome c and other pro-apoptotic factors from the mitochondria, often referred to as mitochondrial outer membrane permeabilization (MOMP), leading to activation of caspases.

Potential applications of hBAX

In our system we would like to induce apoptosis by increasing the amount of proapoptotic proteins such as BAX by producing an expression vector containing TRE-minimal CMV promoter, BAX or PUMA and polyA tail. The proapoptotic proteins are able to express only in a controlled form using the advanced Tet-On expression system. The overexpression of these proteins can lead to Mitochondrial Outer Membrane Permeabilisation (MOMP) which is a point of no return in the mitochondrial pathway of Programmed Cell Death (PCD). The goal is to “switch off” the cells, which were genetically modified by us, after they “finished their work” in the recent local environment combining the components of the tetracycline-inducible system in self-contained retroviral and plasmid vectors.

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NAU-CHINA 2017

The 579bp fragment was cloned under the GAL1 promoter, followed by CYC1 terminator. The pYES2 vector is designed for native expression of protein of interest in S. cerevisiae. It contains the URA3 gene for selection in yeast and 2µ origin for high-copy maintenance.

We can see a significant decrease of yeast concentration . After centrifugation, fewer precipitated cells of induced medium were seen than that of uninduced medium. Yeast growth curve induced by galactose. The expression of Bax protein was activated by inducer-2% galactose. The cells were washed and diluted 1:1000 into fresh medium to induce the GAL promoter. We use the microplate reader to test the value of OD600. By comparing pYES2-Bax’s growth-inhibiting effect with the control, we can see from the picture that the inhibition level of Bax mutant form1 induced by 2% galactose is significantly higher than the control. It is remarkable that expression of the proapoptotic protein Bax conferred a lethal phenotype in the yeast. Yeast growth curve (with glucose as carbon source). In theory, when there is glucose, Bax protein is not expressed. However, from the picture we can be seen that there is a certain leakage. Typan Blue Staining Cell Viability Assay . The stained cells in the view are dead cells. Comparison between two groups was performed using Student's t-test. Results are expressed as mean ± SD of 3 independent experiments. Differences between means were considered significant when the two-tailed P-value was <0.05.. Bax-expressing yeast, the number is getting less and less over time Kill switch The yeasts colonies were observed in a series of time gradients. It is evident that after the induction by galactose, and almost disappeared after 24 hours, while the negative control colony still stayed alive.