Behjat Majidi; Mohsen Fathi Najafi; Shahriar Saeedian
Abstract
Brucella sp. Lumazine synthase, the enzyme involved in riboflavin biosynthesis composed of 10 identical subunits. According to extended applications of LS, it is necessary to set up a high yield expression and purification method for this enzyme.in current study, Lumazine synthase primary structure ...
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Brucella sp. Lumazine synthase, the enzyme involved in riboflavin biosynthesis composed of 10 identical subunits. According to extended applications of LS, it is necessary to set up a high yield expression and purification method for this enzyme.in current study, Lumazine synthase primary structure was achieved from NCBI database and it was expressed by pET28a in BL21 E. coli. The optimum concentrations of IPTG and Kanamycin was evaluated and applied for high yield expression of rBLS. For LPS removal and purification of protein, ammonium precipitation and ion exchange chromatography were performed and no background in ELISA (against Brucella) was observed. ELISA and Western Blotting techniques were applied for expression and purification confirming. For monitoring of purification, SDS-PAGE was applied. Purification of protein with DEAE sephadex (Diethylaminoethyl) resulted in a single band purified rBLS. The approach applied in this study can be used in generation a relatively pure rBLS as a valuable recombinant product in vaccine industries.
