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

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).