The Purification, Crystallization Conditions Screening And The Secondary Structure Analysis Of Harpin_Xooc

Harpin can cause plants to become hypersensitive cell death and induce the growth and defense systems to resist infection of a variety of diseases. Previous studies have demonstrated that there are interactions between Harpin activators and non-host plants, but the mechanism of the interaction is still unknown. To solve these problems and identify the functions of Harpin, studies of its spatial structure are needed. Protein function occurs at the level of the quaternary structure. However, studies of protein structure remain at the level of functional genes.It is difficult to fully understand a protein's function based on its primary or secondary structure, even though the spatial structure can be predicted by sequencing the primary structure. Thus, it is extremely important to understand the biological function and reveal the three-dimensional structure of Harpin. In recent years, methods have improved for the detection of protein spatial structure, e.g., X-ray diffraction analysis. The three dimensional structure of Harpinxooc is particularly important for understanding the way in which Harpin interacts with receptors to induce resistance mechanisms. Growing high-quality protein crystals is an obstacle to obtaining the 3-dimensional structure of proteins by X-ray crystallography.Highly purified Harpinxooc protein is necessary for crystallization. Thus, purification methods for Harpinxooc were established in this study. Harpinxooc was expressed in BL21 cells transformed with a recombinant plasmid, pET-30a-hrf2. Through a series of purification steps, His-select gel column purification and gel-filtration chromatography, highly purified and stable HarpinXooc protein was produced for crystallization. Additionally, the polyclonal antibody of Harpinxooc was prepared by the high purity antigen for further studies of the interactions between Harpinxooc and the non-host plants.In order to study the secondary structure of the protein Harpinxooc and its function domain Harpinxooc (1-94), CD spectra were recorded in the far UV region. Two minima centred around 206nm and 223nm are clearly discernible in the spectrum, suggesting the presence of predominantly ?-helical structure. The content of different types of secondary structures in HarpinXooc and HarpinXooc (1-94) were also estimated:HarpinXooc? ?-helix 45%, ?-sheet 2.3%, ?-turn 9.49%, Unordered 42.1%. HarpinXooc(1-94), ?-helix 40.7%, ?-sheet 7.4%, ?-turn13.8%, Unordered 36.8%. The secondary structure was also predicted from its primary structure using Self-Optimized Prediction Method from Alignments (SOPMA). In order to obtain HarpinXooc crystals of high quality for X-ray crystal diffraction analysis, the stability of HarpinXooc was tested. The results provided a basis for determination of the optimal crystallization conditions. Then HarpinXooc was crystallized with the sitting drop method, using seven kits from Hampton company at different temperature (4?,22?) and different concentrations (1-10 mg/mL).