Author/Authors :
Ghasemi، Amir نويسنده Associate Professor, Operative Dentistry, School of Dentistry, Shahid Behesht University of Medical Science. Shahid Behesht, Iran , , Salari، Mohammad Hossein نويسنده Department of Pathobiology, School of Public Health, Tehran University of Medical Sciences, Tehran, IR Iran , , Pourmand، Mohammad Reza نويسنده Department of Medical Biotechnology, School of Advanced Technology, Tehran University of Medical sciences, Tehran, IR Iran , , Zarnani، Amir Hassan نويسنده Nanobiotechnology Research Center, Avicenna Research Institute, ACECR, Tehran, IR Iran , , Ahmadi، Hojat نويسنده Associte professor of Mechanical Engineering of Agricultural Machinery, University of Tehran, karaj, Iran , , Shirazi، Mohamad Hassan نويسنده Department of Pathobiology, School of Public Health, Tehran University of Medical Sciences, Tehran, IR Iran , , Jeddi Tehrani]، Mahmood نويسنده ,
Abstract :
Background: The development of an effective subunit vaccine against brucellosis is a research area of intense. But optimization of recombinant proteins production in Escherichia coli and content of endotoxins associated with final recombinant proteins are very important.
Objectives: In the present study, expression and purification of Brucella melitensis rHSP and rTF were optimized to reduce endotoxin contaminants.
Materials and Methods: pDEST-tf and pDEST-hsp were transformed into E. coli BL21 (DE3), and then B. melitensis recombinant HSPA and TF proteins were overexpressed. Purification of these proteins was optimized to remove most of endotoxin contaminants from the end product using 0.1% Triton X-114 in washing buffers.
Results: An endotoxin reduction of less than 0.05 EUmg/1 was achieved with protein recovery close to an 80% yield.
Conclusions: As this new protocol requires only one step to simultaneously purify tagged proteins and eliminate endotoxins, it represents a substantial advantage in time, effort, and expense.
