Cloning, Expression, Purification And Crystallization Trials Of Rubber Elongation Factor (REF) From Hevea brasiliensis

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dc.contributor.authorChung, Corrine
dc.date.accessioned2018-01-19T02:14:36Z
dc.date.available2018-01-19T02:14:36Z
dc.date.issued2017-08
dc.description.abstractRubber elongation factor (REF) protein is a protein tightly bound to rubber particles isolated from latex of the rubber tree, Hevea brasiliensis and is well known for its allergenic properties. It is a major component in the biosynthesis of natural rubber but its function is still unclear. It has 137 amino acids with a molecular weight of 14.6 kDa. In this study, an REF gene, namely REF8, was cloned and subsequently the protein products were expressed, purified and used for crystallization trials. REF8 was selected among all the REF isoforms as it is the most commonly reported isoform. The REF8 gene was cloned into the pET SUMO expression vector to construct REF8-pET SUMO. The gene was also codon optimized and added with a 6- histidine tag at the C-ter. It was then cloned at the Nde I and Xho I sites of pET- 22b(+) to construct pET 22b-REF8-6His. The REF8 gene was cloned into these two expression systems in order to obtain soluble REF proteins. The proteins were expressed at different temperatures and IPTG concentrations. However, although highly expressed as a SUMO tagged fusion protein and also as a native protein, REF8 proteins were found to be insoluble in both expression systems. Hence, recombinant SUMO-6His-REF8 and codon optimized REF8-6His were refolded in an attempt to solubilize the proteins. Detergents such as sarkosyl, DDM, OG and HECAMEG were also tested at different concentrations with the hope of obtaining SUMO-6His-REF8 in a soluble form. Both proteins were purified using Immobilized Metal Affinity Chromatography (IMAC) and size exclusion chromatography (SEC). After protein purification, SUMO-6His-REF8 was used for crystallization trials using commercially available crystallization kits. Needle-like crystals were produced at a protein concentration of 93.0 mg/mL in a crystallization solution that contained 0.6 M MgSO4, 0.1 M HEPES sodium pH 7.5, 4% MPD and incubated at 20°C. Further crystallization trials and optimization are being carried out to produce REF protein crystals that are suitable for structural studies.en_US
dc.identifier.urihttp://hdl.handle.net/123456789/5422
dc.language.isoenen_US
dc.publisherUniversiti Sains Malaysiaen_US
dc.subjectCloning, expression, purification and crystallization Trialsen_US
dc.subjectof rubber elongation factoren_US
dc.titleCloning, Expression, Purification And Crystallization Trials Of Rubber Elongation Factor (REF) From Hevea brasiliensisen_US
dc.typeThesisen_US
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