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The recombinant expression and localization of TvCP2 of trichomonas vaginalis

Author

Christopher Keith Wakukawa, University of the Pacific

Date of Award

2012

Document Type

Thesis - Pacific Access Restricted

Degree Name

Master of Science (M.S.)

Department

Biological Sciences

First Advisor

Lisa A. Wrischnik

First Committee Member

Douglas C. Weiser

Second Committee Member

Kirkwood Land

Abstract

Trichomonas vagina/is, one of the most common sexually transmitted diseases, has been shown to increase patients' susceptibility to HIV infection and cervical cancer; moreover, resistance to metronidazole is increasing, and new drug targets must be identified in order to combat resistant strains. T vagina/is expresses cysteine proteases that have been implicated in vaginal epithelial apoptosis as well as immune system evasion. In the past the various cysteine proteases have been studied as a group, and the following work examines, one specific protease, TvCP2, in detail through Western blot analysis, immunofluorescent staining, and recombinant expression. The experiments 5 presented here suggest that aT l-CP2 over-expressing transfectant line processes CP2 and sequesters it in cellular compartments. Previous data gives strong evidence of the secretion of cysteine protease CP4 and hints at the possibility of CP2 secretion as well; however, our results show no co-localization between CP2 and CP4 in T l-CP2 over expressing transfectants, suggesting separate trafficking and different roles. To better characterize CP2 function, we attempted to express active, recombinant protein. Although Pichia pastoris serves as a reliable expression vehicle, a processing event following translation ofTvCP2 appears to have cleaved the pro-domain and, along with it, the a-secretion signal, trapping active TvCP2 within the cellular pellet. A thioreoxintagged version ofTvCP2 has been expressed in E. coli, and preliminary experiments show it may auto-activate under certain conditions, but further experimentation is required to confirm the presence of active CP2 within the fraction purified from these cells.

Pages

65

Recommended Citation

Wakukawa, Christopher Keith. (2012). The recombinant expression and localization of TvCP2 of trichomonas vaginalis. University of the Pacific, Thesis - Pacific Access Restricted. https://scholarlycommons.pacific.edu/uop_etds/813