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:: Volume 17, Issue 4 (Iranian South Medical Journal 2014) ::
Iran South Med J 2014, 17(4): 516-523 Back to browse issues page
Expression, Purfication and endotoxin removal of Brucella melitensis DnaK and Omp31 proteins
Amir Ghasemi1, Mohammed Hussain Salari 2, Amir Hassan Zarnani3, Mahmoud Jeddi-Tehrani4
1- Department of Pathobiology, School of Public Health, Tehran University of Medical Sciences, Tehran, IRAN
2- Department of Pathobiology, School of Public Health, Tehran University of Medical Sciences, Tehran, IRAN , mhsalari2002@gmail.com
3- Nanobiotechnology Research Center, Avicenna Research Institute, ACECR, Tehran, IRAN
Immunology Research Center, Iran University of Medical Sciences, Tehran, IRAN
4- Monoclonal Antibody Research Center, Avicenna Research Institute, ACECR, Tehran, IRAN
Abstract:   (3592 Views)

Background: New strategies are needed to protect against Brucella melitensis infection. Subunit vaccines offer a promising approach because they can stimulate both cellular and humoral immunity, so high production of recombinant protein with less content of endotoxin is desired. In present study, we described a method for expression and purification of B.melitensis recombinant DnaK(rDnaK) and Omp31(rOmp31) proteins while less content of endotoxins were detected in final product. Material and Methods: Recombinant pET-dnak and pDEST-omp31 plasmids were transformed into competent expression host E.coli BL21 (DE3). After induction by IPTG, bacteria were grown at 20◦C for 22h. Then recombinant proteins were purified by Ni-NTA Agarose. Purification was done while two methods using Triton X-114 in washing steps and standard protocol (without detergent) were used in parallel. Results: rDnak and rOmp31 were purified by using Urea. We could obtain 20 and 8 mg recombinant proteins from rDnak and rOmp31 from 1 liter medium, respectively. The amount of endotoxins in final products was less content of 0.05 EU/mg. Furthermore, recovery of protein was up to 80% as compared to the standard protocol. Conclusion: The method used in this study, gives a product with very low extent of endotoxin, but 20% of recombinant proteins were lost. So we think the method described here can be used for purification and endotoxin removal of other recombinant proteins with similar physiologic properties.

Keywords: recombinant protein, purification, endotoxin, expression
Full-Text [PDF 495 kb]   (1730 Downloads)    
Type of Study: Original | Subject: General
Received: 2014/08/11 | Accepted: 2014/08/11 | Published: 2014/08/11
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Ghasemi A, Salari M H, Zarnani A H, Jeddi-Tehrani M. Expression, Purfication and endotoxin removal of Brucella melitensis DnaK and Omp31 proteins. Iran South Med J. 2014; 17 (4) :516-523
URL: http://ismj.bpums.ac.ir/article-1-567-en.html


Volume 17, Issue 4 (Iranian South Medical Journal 2014) Back to browse issues page
دانشگاه علوم پزشکی بوشهر، طب جنوب ISMJ

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