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Studies on the Conformation in Extracellular Domain of Human RANKL by Cystine's Oxidation

Author: ZhangQin
Tutor: PanJiCheng
School: Hubei Normal University,
Course: Analytical Chemistry
Keywords: RANKL prokaryotic expression oxidation polymers conformationalchanges
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Type: Master's thesis
Year: 2013
Downloads: 3
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Abstract


Receptor Activator of Nuclear Factor-κ B Ligand, RANKL, plays an importantrole in a series of physiological processes about osteoclast proliferation,differentiation, activation, survival, tumor cell apoptosis and tumorigenesis, tumormetastasis. In this study, we expressed and purified the extracellular domain of humanRANKL protein, and researched oxygen free radicals affect the RANKL-extracellulardomain, and then regulate the RANKL/RANK signaling pathway.In this article, human RANKL gene were cloned into prokaryotic expressionvector pET-21b and transformed into the expression bacteria E. coli Rosetta. Wesuccessfully constructed recombinant human RANKL gene Rosetta/pET-21b-RANKL.And it was induced to express the target protein. We optimized its expressionconditions. When the IPTG concentration was0.2mmol/mL, the induced timingOD600for0.6, oscillation induced culture for6h in20℃and glycerol concentrationfor4%, efficiently expressed the extracellular domain of human RANKL protein(pET-21b-RANKL) could be acquired in the supernatant. It provided a foundation forfurther purification of the protein and researched its structure function. Crude extractsof human RANKL-extracelluar domain’s protein were isolated and purified by Niaffinity chromatography with10mM,40mM,125mM and250mM gradient ofimidazole elution. SDS-PAGE electrophoresis showed that a electrophoresis pureprotein could be obtained after the40mM imidazole elution. The western blotreference the purified protein to the RANKL-extracelluar domain. Primary structureanalysis and combination of molecular modeling showed that the RANKLextracellular C-terminal domain (amion acids141-317) of176amino acids, whichcontains a cysteine and it at the surface on the position in space. It is speculated thatthere may be oxidized. The intrinsic fluorescence spectrometry, ANS fluorescencespectroscopy, circular dichroism spectroscopy and molecular exclusionchromatography spectroscopy (SEC) were used to detect the effects of oxidizedcystine to structural changes of recombinant human RANKL protein. The results showed that the oxidation leads to the conformational change of RANKL protein andaffected the formation of ploymer. An assumption was made about "oxidation canaffect ploymer on RANKL, thereby affect the formation of hexamer, which ultimatelyaffect the RANKL/RANK signaling pathway ". The oxidation can cause the changesof the protein structure and its function, thus affecting the formation of ploymer. Butthe specific mechanism is unclear and needs further research with a view to provide anew evidence for the RANK/RANKL signaling pathway drug targeting sites,screening small molecule antagonists.

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