Abstract
Apoptosis is the process of programmed cell death. The Bcl-2 family of proteins plays a critical role in the important step of the release of Cytochrome C into the cell. This family of proteins partially consists of BAX and BH3-Only proteins. Previously, there have been many contradictory models of how BAX interacts with BH3-Only proteins to promote or inhibit the apoptotic process. In this research, the BAX-BH3 interaction was examined to shed light on the mechanism. BAX was mutated with L 63 C, T 56 C, and R 109 A mutations in order to introduce instability in the interaction and sites for spin labelling. These mutants created the foundation for the future analysis of the BAX-BH3 Only interaction following protein expression. The BAX DNA which was used was lacking a stop codon, resulting in extra protein being transcribed. BAX protein was also extremely toxic to cells, resulting in the death of cells which were producing large amounts of BAX protein. Due to these problems in the BAX, additional mutations were introduced to include Nde1 and Sap1 cut sites around the BAX DNA sequence. The plasmid containing BAX was digested and separated using Gel Electrophoresis. The attempt was then made to begin ligating the mutant BAX DNA into the pTXB1 vector, a vector which would allow for easier expression and purification of mutant BAX protein. The ligation process was being tested, but remained unsuccessful. This research began the process of optimizing the ligation process. The ligation process concluded this research.