Poster Presentation Melbourne Protein Group Student Symposium 2013

The Binding Activity of Recombinant Plant-made Soluble Receptor for Advanced Glycation Endproducts (sRAGE) to HMGB-1 (#66)

Dinamithra S.R. Premaratne 1 , Mark Mulcair 1 , Raelene J. Pickering 2 , Andrew Sivakumaran 3 , Merlin C. Thomas 2 , Matthew C. Wilce 3 , Diane E. Webster 1
  1. School of Biological Sciences, Monash University, Clayton, VIC, Australia
  2. Biochemistry of Diabetic Complications, Baker IDI Heart and Diabetes Institute, Melbourne, VIC, Australia
  3. School of Biomedical Sciences, Monash University, Clayton, VIC, Australia

The receptor for advanced glycation end-products (RAGE) is a member of the immunoglobin super family of cell surface receptors. Advanced glycation end products (AGEs), Matrix proteins, members of the S100/calgranulin protein family and High Mobility Group family proteins like HMGB-1 are some of the known ligands of RAGE which can play a major role in human disease development and progression. The soluble receptor for advanced glycation endproducts (sRAGE) has the ability to prevent the binding of AGEs to RAGE or other cell surface receptors. sRAGE acts as a dummy receptor by preventing the activation of RAGE and subsequent disease development in humans. Thus sRAGE has potential therapeutic properties which can be applied in vivo. For this protein to be used therapeutically, production and purification of high quality sRAGE is paramount. A plant expression system was selected to produce sRAGE considering its low cost, scalability and safety compared to other systems. sRAGE can be produced with similar patterns of glycosylation as the native RAGE protein. Glycosylation is important for activity and functionality. We successfully expressed sRAGE in hairy roots cultures which was stable and properly glycosylated compared to E. coli made sRAGE. The plant made sRAGE was purified using anion exchange and size exclusion chromatography before its functionality was assessed. The binding ability of plant made sRAGE to HMGB-1 was found to be higher than the purified E. coli made sRAGE which was non glycosylated. The glycosylation of plant made sRAGE is believed to be responsible for this enhanced activity. However, the structure elucidation of plant made sRAGE would further verify this enhanced activity compared to the E. coli made sRAGE in future studies.