Structural Studies Of Allergen Blo T13 From Blomia Tropicalis And KTSC From Methanolobus Vulcani

This disertation mainly contains the following two parts: a structural study of allergen Blo t13(component 13 of tropical dust mite)from Blomia tropicalis and KTSC(Lysine t RNA synthetase C-terminal domain,Lysine is abbreviated as K)from Methanolobus Vulcani.Blomia tropicalis,can cause severe allergic asthma,is a common allergen in tropical and subtropical regions.Blo t13 is a fatty acid-binding protein associated with Ig E mediated allergic reaction.Its epitope induce the production of Ig E,then causes allergy.Although Blo t13 has been identified,the structure of Blo t13 has not been reported at present.In this study,Blo t13 protein was obtained through biological experiments such as molecular cloning and protein purification.High-resolution data of 2 angstroms was obtained by X-ray diffraction,and the crystal structure was sloved by molecular replacement method.The structure is consistent with the fatty acid binding protein family,which consists of 10 ? chains to form a ?-barrel structure,and two ?-helices are located at the end of the ?-barrel.We discussed the structure of Blo t13 and made a preliminary analysis of its potential Ig E binding epitopes,which is critical for understanding of molecular biologial features and development of allergen-specific immunotherapies.A class of small proteins named KTSC exists in Archaea,which function and structure has not been reported.In this study,the KTSC gene of M.Vulcani.was constructed and expressed,the crystal diffraction data of 2.73 ? was collected through purification such as Ni-NTA affinity chromatography and gel filtration chromatography,The tertiary structure was analyzed by molecular replacement method.Each monomer of KTSC consists of 4 ? chains and 2 ? helices.After structural analysis,we found no similarity between the structure of KTSC and the Cterminal domain of lysine t RNA synthase.After searching,we found that the N-terminal domain of KTSC is similar to pyrrolysyl t RNA synthase(pyl RS)N-terminal domain,so we though its name might wrong.Protein translation in organisms is usually limited to 20 standard amino acids.In the case of methanogens,pyl RS use the stop codon UAG to encode pyrrole lysine(Pyl,the natural 22 nd amino acid),which has become a major tool for the expansion of non-natural genetic code in the field of synthetic biology.In addition,the tertiary structure of KTSC determined by us is a tetramer conformation,which is different from previous reports in the literature.By comparing with the N-terminal structure of pyl RS,it is speculated that KTSC could combine RNA.These results provide a new viewpoint for the further study of KTSC family proteins and pyrrolysyl t RNA synthase.