Cloning, Expression, Purification And Crystallization Trials Of Rubber Elongation Factor (REF) From Hevea brasiliensis
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Date
2017-08
Authors
Chung, Corrine
Journal Title
Journal ISSN
Volume Title
Publisher
Universiti Sains Malaysia
Abstract
Rubber 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.
Description
Keywords
Cloning, expression, purification and crystallization Trials , of rubber elongation factor