Expression and purification of the Trypanosoma brucei VDAC in Escherichia coli

The Voltage Dependent Anion‐Selective Channel (VDAC), a transmembrane â‐barrel protein on the mitochondrial outer membrane, is highly conserved among all eukaryotes. VDAC is the most abundant protein on the outer membrane and is responsible for regulating the transport of metabolites that are necessary for respiration and energy production by mitochondria. Trypanosoma brucei is a hemoflagellated protozoan, which is the causative agent of African Trypanosomiasis. T. brucei belongs to the group of earliest eukaryotes and possesses VDAC, which is similar in structure with bacterial outer membrane porin Omp85. The objective of this study is to determine whether the recombinant T. brucei VDAC (rTbVDAC) can function as a selective anion channel when reconstituted in an artificial membrane. To obtain the purified recombinant protein, we cloned the ORF for TbVDAC in a bacterial expression vector, pQE30. The protein was expressed in E. coli after induction with IPTG (1 mM). The expression conditions were optimized for maximum production. The rTbVDAC was solubilized under native conditions and subjected to purification by Nickel‐agarose chromatography. Currently, we are optimizing the purification process to obtain a sufficient quantity of purified protein that will be used for subsequent electrophysiological studies to determine to what extent this recombinant protein can function as VDAC.