Date of Award
Honors College Thesis
Hao Xu, Ph.D.
Acting as the chief mediators of vesicular fusion, soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) play a role in many intracellular trafficking events by moving opposing membranes into close proximity. One such event takes place in the process of autophagy. A key SNARE involved in autophagy is Synaptosome Associated Protein 29 (SNAP-29), which acts on the autophagosome membrane to promote autophagosome and lysosome fusion. Kaposi’s Sarcoma Herpesvirus (KSHV) proteins ORF33 and ORF38 were demonstrated to interact with SNAP-29. The exact mechanism of this interaction is yet to be elucidated but it is hypothesized that these interactions allow KSHV to modulate mast cell autophagy to sustain viral replication. Mast cell degranulation was also demonstrated to be increased upon exposure to KSHV, which might increase tumor progression and viral replication in Kaposi’s Sarcoma. To fully characterize the interaction between SNAP29 and KSHV proteins, a recombinant pMBP-parallel-1 expression vector containing human SNAP-29 cDNA was generated. PCR was utilized to amplify SNAP-29 cDNA and insert restriction enzyme sites. Restriction digestion and subsequent ligation were carried out to generate the recombinant pMBP-parallel-1-SNAP-29 construct. This construct was confirmed by sequencing and then used to transform Rosetta 2 (DE3) E. coli cells. These cells were then induced by IPTG to express the SNAP-29 protein. SNAP-29 expression was confirmed by the increased intensity of the 71.5 Kda band on an SDS-PAGE gel. Future site-directed mutagenesis of the generated SNAP-29 construct will allow for the identification of the functional domains within SNAP-29 that interact with the ORF33/38 complex through lipid-mixing assays. It is anticipated that these studies may reveal potential therapeutic targets for Kaposi’s Sarcoma.
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Ryals, Logan M., "Cloning and Expression of Human Synaptosome Associated Protein 29 in E. coli" (2020). Honors Theses. 744.