Cloning, expression, purification and structure determination of peanut allergen Ara h 5.

摘要

In our study, the peanut allergen Ara h 5 was cloned from raw peanut mRNA. The cDNA of the gene was then introduced into different expression vectors for protein expression in different E.coli strains. Recombinant protein expression was very successful with useful amounts of soluble protein produced. Fast protein liquid chromatography was used to purify the recombinant Ara h 5. High purity protein was subjected to crystallization screen and good quality crystals were harvested. Several crystallographic data sets were collected at a synchrotron X-ray beam line. The three-dimensional structure of the peanut profilin Ara h 5 was determined to 1.10A resolution. The purified protein and the purification methods can be used in future research on the protein's allergenecity, cross-reactivity and allergy immunotherapies. The high resolution structure was compared with the structures of homologous allergens and the putative epitopes was displayed on the allergen structure to evaluate their possible legitimacies. In the future, the Ara h 5 structure could be very valuable in studies of allergenecity and in the design of future immunotherapies.