Volume 5, Issue 2 (Vol.5 No.1 Apr 2017)                   rbmb.net 2017, 5(2): 103-107 | Back to browse issues page

XML Print

Download citation:
BibTeX | RIS | EndNote | Medlars | ProCite | Reference Manager | RefWorks
Send citation to:

Mahmoodi M, Ghodsi M, Moghadam M, Sankian M. Pulsed Dilution Method for the Recovery of Aggregated Mouse TNF-α. rbmb.net. 2017; 5 (2) :103-107
URL: http://rbmb.net/article-1-109-en.html
Immuno-Biochemistry lab, Immunology Research Center, Buali Research Institute , School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran.
Abstract:   (9187 Views)

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 19.9 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 62.5 µg/mL.

Conclusions: Our study demonstrates the efficiency of this method to produce a high yield of folded mature TNF- (α).

Full-Text [PDF 422 kb]   (2448 Downloads)    
Type of Article: Original Article | Subject: Cell Biology
Received: 2016/06/3 | Accepted: 2016/07/20 | Published: 2017/04/30

Add your comments about this article : Your username or Email:

Send email to the article author

Rights and permissions
Creative Commons License This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.

© 2015 All Rights Reserved | Reports of Biochemistry and Molecular Biology

Designed & Developed by : Yektaweb