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dc.contributor.advisor Lin, Wei-jen en
dc.contributor.author Chatrathip, Nasongkla en
dc.date.accessioned 2018-05-08T20:46:08Z en
dc.date.available 2018-05-08T20:46:08Z en
dc.date.issued 2018-05-08 en
dc.identifier.uri http://hdl.handle.net/10211.3/202533 en
dc.description.abstract Botulism is a fatal disease caused by the botulinum neurotoxin (BoNT), a lethal exotoxin affecting the peripheral nervous system leading to descending flaccid paralysis and respiratory failure. BoNT exists as part of a progenitor toxin complex (PTC) as produced by Clostridia spp. Members of the BoNT serotype A1 produce PTCs containing the BoNT and associated non-toxic proteins (ANTPs). ANTPs consist of a non-toxic nonhemagglutinin protein and a hemagglutinin (HA) complex—HA17, HA33, and HA70, which is postranslationally cleaved into HA52 and HA19. Our Yeast Two Hybrid study has shown the interaction of recombinant HA17 with the C-terminus of recombinant HA52, designated rHA52-5 (corresponding to HA70 residues Met508-Asn626) of the botulinum neurotoxin (BoNT) complex serotype A1. This is in agreement with the molecular arrangement resolved by the crystal structure studies. The objective of this study is to provide further in vitro confirmation of this interaction by pull-down analysis of recombinant HA proteins. We hypothesized that (1) rHA52-5 and rHA17 can bind to each other in vitro, and (2) mutations of the amino acids involved in interaction between rHA52-5 and HA17 based on the crystal structure will weaken their association in vitro. To verify this, the hemagglutinin genes of Clostridium botulinum Hall A were cloned into expression vectors to produce recombinant proteins. Alanine screening through site-directed mutagenesis was performed on rHA17 to analyze the contribution of specific residues involved in hydrogen bonding between HA17 and HA70D3 (residues Pro378-Asn626) based on the crystal structure. Pull down analysis showed positive in vitro interaction between rHA17 and rHA52-5, supporting the Yeast Two Hybrid system and crystal structure molecular arrangement. Significant differences in binding affinity in the pull-down assay among the wild-type and variants showed that certain residues have influence in the binding affinity. Additionally, double mutations of HA17 can play a more significant role due to the requirement of multiple bonds to maintain this interaction It has been reported that the entire HA complex is necessary for successful transcytosis of PTCs through the epithelial M cells and eventual arrival in the neuromuscular junction. Therefore, understanding the interactions among the HAs will provide insight to approaches in disrupting these interactions and preventing botulism. en
dc.format.extent 103 pgs. en
dc.language.iso en en
dc.publisher California State Polytechnic University, Pomona en
dc.rights.uri http://www.cpp.edu/~broncoscholar/rightsreserved.html en
dc.subject botulism en
dc.subject Clostridium botulinum en
dc.subject hemagglutinin en
dc.subject HA17 en
dc.subject HA52 en
dc.subject HA19 en
dc.subject botulinum neurotoxin complex en
dc.subject botulinum neurotoxin en
dc.subject BoNT en
dc.subject recombinant protein en
dc.title The Study of Interactions Between Recombinant Non-Toxic Associated Proteins HA 17 and the C-Terminus of HA52 of the Botulinum Neurotoxin Complex en
dc.type Thesis en
dc.contributor.department Department of Biological Sciences en
dc.description.degree M.S. en
dc.contributor.committeeMember Snyder, Jamie en
dc.contributor.committeeMember Buckley, Nancy en
dc.rights.license All rights reserved en


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