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'); Reports of Biochemistry and Molecular Biology rbmb.net Basic Sciences http://rbmb.net 1 admin 2322-3480 2322-3480 10.61882/rbmb en jalali 1396 2 1 gregorian 2017 5 1 5 2 online 1 fulltext
en Pulsed Dilution Method for the Recovery of Aggregated Mouse TNF-α زیست شناسی سلولی Cell Biology مقالات اصلی Original Article <p style="text-align: justify;"><strong><em>Background:</em></strong> The expression of mouse tumor necrosis factor alpha (TNF-&alpha;) in <em>Escherichia coli</em> is a favorable way to get high yield of protein; however, the formation of cytoplasmic inclusion bodies, which is the consequence of insoluble accumulated proteins, is a major obstacle in this system. To overcome this obstacle, we used a pulsed dilution method to convert the product to its native conformation.</p> <p style="text-align: justify;"><strong><em>Methods:</em></strong> Reducing agent and guanidine hydrochloride were used to solubilize inclusion bodies formed after TNF-(&alpha;) expression. Then, the refolding procedure was performed by pulsed dilution of the denatured protein into a refolding buffer. The properly-folded protein was purified by metal affinity chromatography.</p> <p style="text-align: justify;"><strong><em>Results:</em></strong> SDS-PAGE showed a 19.9 kDa band related to the mature TNF-(&alpha;) protein. The protein was recognized by anti-mouse TNF-(&alpha;) on western blots. The final concentration of the purified recombinant TNF-(&alpha;) was 62.5 &micro;g/mL.</p> <p style="text-align: justify;"><strong><em>Conclusions:</em></strong> Our study demonstrates the efficiency of this method to produce a high yield of folded mature TNF- (&alpha;).</p> Escherichia coli, Guanidine Hydrochloride, Inclusion Bodies, Mouse TNF-(α) 103 107 http://rbmb.net/browse.php?a_code=A-10-1-73&slc_lang=en&sid=1 Merat Mahmoodi 10031947532846006223 10031947532846006223 No Immuno-Biochemistry lab, Immunology Research Center, Mashhad University of medical Sciences, Mashhad, Iran. Maryam Ghodsi 10031947532846006224 10031947532846006224 No Immuno-Biochemistry lab, Immunology Research Center, Mashhad University of medical Sciences, Mashhad, Iran. Malihe Moghadam 10031947532846006225 10031947532846006225 No Immuno-Biochemistry lab, Immunology Research Center, Mashhad University of medical Sciences, Mashhad, Iran. Mojtaba Sankian sankianm@mums.ac.ir 10031947532846006226 10031947532846006226 Yes Immuno-Biochemistry lab, Immunology Research Center, Buali Research Institute , School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran.