Expression and purification of HIV-I TAT protein transduction domain fused with acid β-glucosidase and enhanced green fluorescent protein

dc.contributor.authorVaags, Andrea Kathleen.en_US
dc.contributor.supervisorChoy, Francis Y. M.en_US
dc.date.accessioned2008-04-10T05:58:07Z
dc.date.available2008-04-10T05:58:07Z
dc.date.copyright2004en_US
dc.date.issued2008-04-10T05:58:07Z
dc.degree.departmentDept. of Biologyen_US
dc.description.abstractThe protein transduction domain (PTD) of the HIV-I TAT protein has been shown to be capable of crossing cellular membranes and even the blood-brain barrier while carrying cargo molecules along with it. Exploiting this property to deliver biologically active acid β-glucosidase (GBA) would be of use to improve the current treatment of Gaucher disease by enzyme replacement therapy. Genetic fusion of the TAT PTD to GBA was performed and the resulting gene was inserted into an insect expression vector, p2ZOptcxF, to allow for heterologous protein production in Sf9 cells. A TAT fusion with enhanced green fluorescent protein (EGFP) was also created to serve as a control. The insect vector encoded a cellulose-binding domain to allow for affinity purification of the heterologous proteins. The Sf9 system produced 0.9-1.2 μg/ml quantities of EGFP fusion proteins, but only low ng/ml levels of GBA fusions. The addition of the cellulose binding domain decreased protein expression, but could be used for purification when μg/ml quantities of protein were produced. This suggests that expression of soluble, unglycosylated proteins such as EGFP can be achieved in the p2ZOptcxF/Sf9 system and to a lesser extent complex, highly-glycosylated proteins such as GBA can also be produced. In order to improve the expression of GBA fusions further optimization of the vector, selection and production must be undertaken.en_US
dc.identifier.urihttp://hdl.handle.net/1828/469
dc.subject.lcshGaucher's diseaseen_US
dc.titleExpression and purification of HIV-I TAT protein transduction domain fused with acid β-glucosidase and enhanced green fluorescent proteinen_US

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