Characterization of TbCatL Recombinant Protein in a CHO-S Mammalian Model
1Department of Biological Sciences, Hunter College; 2Biology PhD Program, The Graduate Center, The City University of New York, New York, NY
Trypanosoma brucei, known to cause African Trypanosomiasis in the sub-Saharan populations of Africa, is a eukaryotic parasite that killed many people. Humans have been able to develop a resistance to this affliction with a trypanolytic lipoprotein, apolipoprotein-1 (APOL1) G0, that forms ion channels in the parasite membranes and causes lysis. However, this never-ending battle continues with the Trypanosoma brucei Cathepsin-L (TbCatL), a protease, is hypothesized to degrade APOL1 G0 and thus allows for the evasion of trypanolysis.
To test this hypothesis, pure recombinant TbCatL will be incubated with rAPOL1 embedded in an artificial lipid bilayer, which mimics the membranes of the parasite. The rAPOL1 will be activated, which results in open channel conformation and ion flux. If rTbCatL can degrade APOL1 in the bilayer, we hypothesize that the ion flux will stop. The transformation of Chinese Hamster Ovary suspension cells (CHO-S) expression with a pcDNA/TbCatL vector will provide rTbCatL needed for experimentation, generating high quality and functional protease that can be purified via proteolytic activation. Work to optimize this process to obtain purer rTbCatL is underway.
Using this novel expression system, we will evaluate if rTbCatL can degrade and inactivate rAPOL1 G0 channels in membranes. With the model, further investigations can be made into how rTbCatL interacts with APOL1 G1 and G2 variants, new participants of the escalated immune arms race, that display a resistance to Trypanosoma brucei that utilize the protease to evade trypanolysis but to the detriment of renal function and health in human African populations.
