2012 Annual Report 1a.Objectives (from AD-416): To advance the fundamental knowledge regarding structure/property relationships in natural rubber, especially the role of naturally-occurring, non-rubber constituents in creating and controlling the nanostructured features that are believed to be responsible for the unique properties of natural rubber derived from Hevea brasiliensis. 1b.Approach (from AD-416): 1) Deconstruct natural rubber latex through selective separation of naturally-occurring proteins and lipids by: a) identifying highly abundant natural rubber proteins (proteomics) b) extract and concentrate proteins from natural latex, including Hevea c) produce recombinant rubber particle proteins 2) Systematically reconstruct the latex composition from synthetic and Hevea, guayule, and Russian dandelion natural latex by protein add-back compositions. 3) Fully characterize the nanostructure and properties of resulting materials. 3.Progress Report: This research is related to parent research Objective 2: Develop technologies to enable the domestic production of natural rubber and ricinoleate with properties as good as or better than existing sources for industrial applications. Identify chemical processes and/or modifications to develop superior performance properties of natural rubber from domestic sources. In FY2012 collaborative research established that addition of amino acids to guayule latex improve the thermo-oxidative stability, regardless of the R functional group. In other words, the peptide bond nature, more specifically, the N from the peptide bond, may participate in rubber stabilization. Separately, expression and purification of Hevea REF (a 14 KDa. protein) and SRPP proteins were carried out using a pGEX-6P-1 vector transferred to E. coli competent cells (Rossetta cells), incubation time was optimized, and the protein purified according to the purification protocols of GST fusion proteins. Unfortunately the protein concentration was lower than desired (about 1.1mg per 500 ml culture). Using a High Cell Density method did not show significant increase in protein expression for both REF and SRPP. It is possible that REF and SRPP in high concentrations have toxic effects on bacterial cell. Our next step will be to try a baculovirus system for cloning and high-level expression of recombinant proteins, using Gateway Technology. In this case the recombinant baculovirus will be used to directly transfect insect cells, which may be less sensitive to high levels of recombinant protein.