title: Pulsed Dilution Method for the Recovery of Aggregated Mouse TNF-α.
abstract: BACKGROUND
  The expression of mouse tumor necrosis factor alpha (TNF-α) inEscherichia coliis 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).
authors.affiliation: Immuno-Biochemistry lab, Immunology Research Center, Mashhad University of medical Sciences, Mashhad, Iran.
authors.forename: Merat
authors.initials: M
authors.lastname: Mahmoodi
authors.affiliation: Immuno-Biochemistry lab, Immunology Research Center, Mashhad University of medical Sciences, Mashhad, Iran.
authors.forename: Maryam
authors.initials: M
authors.lastname: Ghodsi
authors.affiliation: Immuno-Biochemistry lab, Immunology Research Center, Mashhad University of medical Sciences, Mashhad, Iran.
authors.forename: Malihe
authors.initials: M
authors.lastname: Moghadam
authors.affiliation: Immuno-Biochemistry lab, Immunology Research Center, Buali Research Institute , School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran.
authors.forename: Mojtaba
authors.initials: M
authors.lastname: Sankian
keywords: Escherichia coli; Guanidine Hydrochloride; Inclusion Bodies; Mouse TNF-(α) 