The expression of alpha-N-acetylglucosaminidase in two heterologous gene expression systems
| dc.contributor.author | Crawford, Joanna | |
| dc.contributor.supervisor | Choy, Francis | |
| dc.date.accessioned | 2007-12-17T17:23:51Z | |
| dc.date.available | 2007-12-17T17:23:51Z | |
| dc.date.copyright | 2007 | en_US |
| dc.date.issued | 2007-12-17T17:23:51Z | |
| dc.degree.department | Dept. of Biology | en_US |
| dc.degree.level | Master of Science M.Sc. | en_US |
| dc.description.abstract | Mucopolysaccharidosis (MPS) IIIB is an autosomal recessive disorder caused by a defect in alpha-N-acetylglucosaminidase (NAGLU), a lysosomal enzyme involved in the degradation of heparan sulphate. Dysfunctional NAGLU gives rise to a clinical phenotype of severe and progressive mental retardation, often accompanied by hyperactivity and aggressive behaviour. At present, there is no effective treatment for MPS IIIB. However, cloning of the human NAGLU cDNA has made the potential production of human recombinant enzyme for use in enzyme replacement therapy (ERT) a viable option. The work outlined herein focuses on attempts to produce human recombinant NAGLU (rNAGLU) using both yeast and insect cell based expression systems; with the major focus on yeast based expression. Use of a humanized yeast strain, codon optimisation of a portion of the NAGLU gene, selection of Mut+, MutS and multiple integrant strains, and growth at decreased temperature were explored to optimise NAGLU expression in the methylotrophic yeast, Pichia pastoris. As none of these measures resulted in abundant NAGLU production, Sf9 and Tni insect cell lines were investigated as an alternate expression system. Additionally, a protein transduction domain (PTD) was fused to NAGLU (NTAT) to circumvent current problems faced in delivering therapeutic enzymes to the brain. NAGLU protein, with and without a fused PTD, were expressed using stable transfection and baculovirus infection techniques. Small scale experiments utilizing the baculovirus expression vector system (BEVS) have yielded promising results, generating functionally active NAGLU and NTAT protein of the expected approximately 80-85 kDa molecular mass. This preliminary success indicates the BEVS may be an attractive option for the large scale production of rNAGLU and rNTAT. | en_US |
| dc.identifier.uri | http://hdl.handle.net/1828/276 | |
| dc.language | English | eng |
| dc.language.iso | en | en_US |
| dc.rights | Available to the World Wide Web | en_US |
| dc.subject | recombinant gene expression | en_US |
| dc.subject | alpha-N-acetylglucosaminidase | en_US |
| dc.subject | Pichia pastoris | en_US |
| dc.subject | baculovirus expression in insect cells | en_US |
| dc.subject.lcsh | UVic Subject Index::Sciences and Engineering::Biology | en_US |
| dc.title | The expression of alpha-N-acetylglucosaminidase in two heterologous gene expression systems | en_US |
| dc.type | Thesis | en_US |