Patil, Nilesh Baliram (2016) Creating recombinant fusion protein conjugates for targeting SNARE protease into neuroendocrine cells. Master of Science thesis, Dublin City University.
Abstract
Chronic pain poses major healthcare and economic burdens. The aim of this study was to develop a strategy for pain management by generating a biotherapeutic that requires targeting of soluble N-ethylmaleimide-sensitive fusion protein (NSF) attachment protein receptor (SNARE) within the hyper-active sensory nerves. This entailed conjugating nerve growth factor (NGF) and the SNARE-cleaving protease and internalization domains of botulinum neurotoxin A (BoNT/A). The methodology involved genetic fusion and use of a protein stapling technology with a view to replacing the BoNT/A C-terminal neuronal binding subdomain (HCC) with βNGF which is a specific ligand for preferential sensory neuronal targeting. A first generation of BoNT/AΔHCC fused to βNGF was expressed in E. coli and purified. An alternative strategy was also adopted with a view to stapling a recombinant BoNT/AΔHCC-SNAP-25 protein (without the neuronal binding domain) with Vamp2.βNGF produced in E. coli, and a synthetic syntaxin-1 peptide via SNARE complex formation. Treatment of PC-12 cells with BoNT/AΔHCC fused to βNGF did not give any cleavage of intracellular SNAP-25. Failure to redirect BoNT/A protease into PC-12 cells was attributed to inactivity of the βNGF producing E. coli. After a number of attempts, the precursor peptide (Pre-Pro signal peptide) of NGF was identified as an essential element in making a biologically-active βNGF. Encouragingly, after inclusion of this leader peptide sequence into a Vamp2.βNGF construct, it was expressed as an active protein, although with low yield. The latter was addressed by creating a construct encoding a Pre-Pro signal peptide followed by βNGF.Vamp2 in the hope of increasing the yield. Expression of the resultant protein was not attempted. In summary, expression of functionally-active βNGF.Vamp2 was achieved. Further optimization is needed before its conjugation to the BoNT core-therapeutic domains, for redirecting SNARE-cleaving protease into hyper-active sensory nerves.
Metadata
Item Type: | Thesis (Master of Science) |
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Date of Award: | November 2016 |
Refereed: | No |
Supervisor(s): | Dolly, Oliver, Lawrence, Gary and Wang, Jiafu |
Uncontrolled Keywords: | Chronic pain; N-ethylmaleimide-sensitive fusion protein; NSF; SNARE |
Subjects: | Biological Sciences > Neuroscience Humanities > Biological Sciences > Neuroscience |
DCU Faculties and Centres: | UNSPECIFIED |
Use License: | This item is licensed under a Creative Commons Attribution-NonCommercial-No Derivative Works 3.0 License. View License |
Funders: | Science Foundation Ireland |
ID Code: | 21388 |
Deposited On: | 17 Nov 2016 11:32 by Oliver Dolly . Last Modified 19 Jul 2018 15:09 |
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