Pulsed Dilution Method for the Recovery of Aggregated Mouse TNF-α.
Year: 2017
Type: article
Abstract: BACKGROUND: 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).
Source: PubMed
Authors: Merat Mahmoodi, Maryam Ghodsi, Malihe Moghadam, Mojtaba Sankian
Institution: Mashhad University of Medical Sciences
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W2560828720