The expression of alpha-N-acetylglucosaminidase in two heterologous gene expression systems

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dc.contributor.author Crawford, Joanna
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.identifier.uri http://hdl.handle.net/1828/276
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.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
dc.contributor.supervisor Choy, Francis
dc.degree.department Dept. of Biology en_US
dc.degree.level Master of Science M.Sc. en_US

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