Date of Award
Santa Clara : Santa Clara University, 2020.
Master of Science (MS)
The mechanism of Sortase A substrate specificity has been widely studied and applied to many approaches to bioconjugation. Current research includes transpeptidation between peptide nucleotide acids, polypeptides, viruses, or antibodies. These applications help improve drug targeting and delivery. Scientists have performed Sortase A-mediated protein ligation in vitro. This project proposes an in vivo protein ligation method with Sortase A. In this design, Sortase A acts as a catalyst to initiate bioconjugation between the LPETG motif and pentaglycine (Gly5) chain to express GFP. This technique bypasses the ribosome and offers an alternative way to synthesize protein in mammalian cells. Three recombinant plasmids were created with the GFP gene spliced into two segments. The LPETG motif is inserted to the N-terminus of GFP and the Gly5 chain is attached to the C-terminus of GFP. The recombinant plasmids are co-transfected into HeLa cells with another recombinant plasmids that expresses only for Sortase A. The expression of GFP is observed under a fluorescence microscope every 12 hours. Detection of GFP by fluorescence indicates the reconstruction of whole GFP via in vivo protein ligation is successful. Overall, this design pioneers synthetic biology by reengineering cellular pathways in a mammalian cell. There is potential to create synthetic cells and apply Sortase A to trigger in vivo ligation between a protein drug and a drug target for precision medicine.
Choppa, Akhil, "In Vivo Non-Ribosomal Protein Synthesis in Mammalian Cells" (2020). Bioengineering Master's Theses. 9.