Construction of CNFY-CNF1 Chimera Protein for Study of Differential pH Dependent Activity of CNF Proteins

toxin;dermonecrotic;cytotoxic necrotizing factors (CNF);chimera;translocation;pH dependent

Chan, Philemon ; Wilson ; Brenda A.

关键词: toxin;    dermonecrotic;    cytotoxic necrotizing factors (CNF);    chimera;    translocation;    pH dependent;   

Others  :  https://www.ideals.illinois.edu/bitstream/handle/2142/49697/Philemon_Chan.pdf?sequence=1&isAllowed=y

美国|英语

来源: The Illinois Digital Environment for Access to Learning and Scholarship

【 摘 要 】

In this thesis we will explore the topic of cytotoxic necrotizing factors (CNFs) and show the creation of chimeric constructs to further test the details of CNF translocation into the cell. The CNF family of toxins is under the larger family of dermonecrotic AB toxins. These AB toxins have the ability to cause dermonecrosis when applied to the skin of animals. Among the dermonecrotic toxins, Pasteurella multocida toxin (PMT) and cytotoxic necrotizing factors 1, 2, and Y (CNF1, 2, and Y) are of interest. PMT, CNF1, CNF2, and CNFY are closely related in amino acid sequence. In a previous study on PMT, the method of translocation of the toxin into the cell for the release of the active domain was studied. Results showed that PMT enters the cell through back-trafficking in the endosomal pathway. It was also found that CNFs follow this same pathway into the cell. Interestingly, CNF1 and CNF2 were found to behave with increased efficiency of translocation across the endosomes when subjected to low levels of the inhibitors bafilomycin A1 (BafA1) and ammonium chloride (NH4Cl) compared with CNFY and PMT. A sequence comparison showed that CNF1 and CNF2 have higher pI in a specific region on the translocation domain than that of CNFY and PMT; a probable cause of the difference in translocation under low inhibitor levels. This being so, an experiment to specifically isolate and test the differences in activity caused by the translocation region was proposed. Chimeric CNF constructs involving variable translocation domains among the CNFs with constant activity domain and vice versa are proposed. In this thesis we specifically discuss the construction of the CNFY1 chimera from CNFY translocation domain and CNF1 activity domain. Generation of this chimera provides a foundation for additional constructs, as well as direction to begin testing. Using serum response element (SRE) has been seen as an effective way to test cellular activity for PMT and CNFs. In this thesis, we focus on the construction of CNF chimeras so that SRE-based reporter tests may be conducted in future studies.