Expression, Purification And Crystallization Trials Of Small Rubber Particle Protein (Srpp) From Hevea Brasiliensis
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Date
2016-02
Authors
Satinder Singh, Saranpal Singh
Journal Title
Journal ISSN
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Publisher
Universiti Sains Malaysia
Abstract
Natural rubber (NR) of Hevea brasiliensis predominantly made up of cis-1,4-polyisoprene, is an essential industrial commodity with unique characteristics. The biosynthesis of NR from H. brasiliensis is catalyzed on the surface of rubber particles by a set of integrated proteins, namely the small rubber particle protein (SRPP), and rubber elongation factor (REF). The SRPP protein has been speculated to incorporate isopentenyl diphosphate (IPP) monomers in rubber particles. Furthermore, the SRPP protein has also been implicated as a major latex allergen. Though evidence suggests the involvement of these proteins in rubber biosynthesis and as an allergen, there is still a dearth of research pertaining to the mechanism that supports the association of this protein onto the rubber particles and mechanism of allergenic disease. Hence, this research aims to clone, express, purify and thereafter to perform crystallization trials on the purified SRPP protein. Briefly, the codon optimized vector designated as SRPP_His was expressed in E. coli BL21 (DE3) cells. The SRPP_His protein of 23 kDa expressed in the form of inclusion bodies was then denatured, purified by immobilized metal affinity chromatography (IMAC) and refolded in the presence of 1 M arginine hydrochloride. Using size-exclusion chromatography (SEC) coupled with Dynamic Light Scattering (DLS), SRPP_His was seen to form higher order oligomers with high polydispersity (~35%), which could be a result of unspecific hydrophobic interactions. Hence, several sets of detergents were tested in the hope to obtain SRPP_His in a homogenous form. In addition, NusA_SRPP fusion construct was designed parallelly to readily express the SRPP protein in a soluble form. Using 0.2% (w/v) of sarkosyl, the SRPP_His was solubilized and purified by IMAC and SEC. Surprisingly, even in the presence of sarkosyl, the SRPP_His remained as a soluble oligomer. Further investigations by far-UV circular dichroism (CD) spectroscopy revealed that SRPP_His solubilized in sarkosyl is partially unfolded. As for NusA_SRPP, the protein was observed to elute as tetramers and a dimers. During crystallization screening, tiny clustered needle crystals were formed in solution #45 of Crystal screen 1 and solution #27 of Crystal screen 2 for SRPP_His solubilized in sarkosyl. However, using control experiments, it was shown that the needle-like crystals were merely salt crystals, and not protein crystals. For SRPP_His in arginine hydrochloride and NusA_SRPP, preliminary crystallization trials resulted in protein precipitation. Different approaches are necessary such as Lipidic Cubic Phase (LCP) method in order to crystallize SRPP_His. Structural studies of SRPP will provide an understanding on the specific mechanism that is involved in the rubber biosynthesis and allerginicity.
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Keywords
Rubber