Mucopolysaccharidosis IIIB (MPS IIIB) is an autosomal recessive disorder of glycosaminoglycan (GAG) metabolism. Disruption of the gene encoding a-N-acetylglucosaminidase (Naglu) results in the inability to degrade the GAG heparan sulfate (HS). Consequently. undegraded HS builds up and results in the secondary accumulation of gangliosides and substantial changes in the expression of genes related to neural cell growth and function. Clinically, affected individuals display hyperactivity. insomnia and severe and progressive mental retardation. Currently. no treatment or cure is available for this devastating disorder which is ultimately fatal. Enzyme replacement therapy is one method being examined as an avenue for treatment of MPS IIIB. but it has yet to overcome difficult obstacles, such as production and targeted delivery. This thesis examines the use of the methylotrophic yeast Pichia pastoris as a host for the production of recombinant Naglu. A protein transduction domain (PTD) derived from the HIV-l Tat protein was fused to Naglu to circumvent the current problems faced in delivering this therapeutic enzyme. Expression of this fusion protein was tested in four different strains of Pichia. each with unique attributes. Though the Naglu produced was in an active recombinant form. it was not abundant and this has precluded further characterization. It is likely that inefficiency at the transcriptional/post-transcriptional level hindered higher expression levels. Optimization of these factors may well facilitate Naglu expression in Pichia pastoris. and ultimately allow for substantial enzyme production for use in replacement therapy.
| Identifer | oai:union.ndltd.org:uvic.ca/oai:dspace.library.uvic.ca:1828/1869 |
| Date | January 2006 |
| Creators | Patrick, Chelsea Marie |
| Contributors | Choy, Francis |
| Source Sets | University of Victoria |
| Language | English, English |
| Detected Language | English |
| Type | Thesis |
| Rights | Available to the World Wide Web |
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