Pulsed Dilution Method for the Recovery of Aggregated Mouse TNF-α

Publish Year: 1396
نوع سند: مقاله ژورنالی
زبان: English
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شناسه ملی سند علمی:

JR_RBMB-5-2_005

تاریخ نمایه سازی: 10 شهریور 1400

Abstract:

Background: The expression of mouse tumor necrosis factor alpha (TNF-α) in Escherichia coli 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. Methods: Reducing agent and guanidine hydrochloride were used to solubilize inclusion bodies formed after TNF-(α) 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. Results: SDS-PAGE showed a ۱۹.۹ kDa band related to the mature TNF-(α) protein. The protein was recognized by anti-mouse TNF-(α) on western blots. The final concentration of the purified recombinant TNF-(α) was ۶۲.۵ µg/mL. Conclusions: Our study demonstrates the efficiency of this method to produce a high yield of folded mature TNF- (α).

Authors

Merat Mahmoodi

Immuno-Biochemistry lab, Immunology Research Center, Mashhad University of medical Sciences, Mashhad, Iran.

Maryam Ghodsi

Immuno-Biochemistry lab, Immunology Research Center, Mashhad University of medical Sciences, Mashhad, Iran.

Malihe Moghadam

Immuno-Biochemistry lab, Immunology Research Center, Mashhad University of medical Sciences, Mashhad, Iran.

Mojtaba Sankian

Immuno-Biochemistry lab, Immunology Research Center, Buali Research Institute , School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran.