Gaucher disease (GD) is caused by an inherited deficiency of the human lysosomal enzyme acid β-glucosidase (GBA, EC 3.2.1.45). Absence of functional enzyme results in lysosomal glycolipid accumulation. This disorder primarily affects organs of the reticuloendothelial system and disease severity ranges from mild hepatosplenomegaly to extreme neurological degeneration. Disease symptoms have been shown to be greatly ameliorated by enzyme replacement therapy (ERT). Limitations to therapy include the high cost of current ERT and its inability to treat neurological symptoms. In the present study I sought to produce a GBA-fusion enzyme in an economical manner that can be used to treat neurological GD. I explored the use of Pichia pastoris as an economical recombinant protein expression system for production of human GBA. In addition, I synthesized a protein transduction domain (PTD)-GBA fusion protein for its potential to be used as a neurotherapeutic. The results show that GBA-PTD4 can be expressed and purified from P. pastoris. Hydrophobic interaction chromatography and gel filtration chromatography were successful in purifying GBA-PTD4. Further optimization of expression and purification techniques is required for effective large scale production of recombinant enzyme. / Graduate
| Identifer | oai:union.ndltd.org:uvic.ca/oai:dspace.library.uvic.ca:1828/3344 |
| Date | 02 June 2011 |
| Creators | Goebl, April Mary |
| Contributors | Choy, Francis Y. M. |
| Source Sets | University of Victoria |
| Language | English, English |
| Detected Language | English |
| Type | Thesis |
| Rights | Available to the World Wide Web |
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