title: Refolding process of cysteine-rich proteins:Chitinase as a model.
abstract: BACKGROUND
  Recombinant proteins overexpressed in E. coli are usually deposited in inclusion bodies. Cysteines in the protein contribute to this process. Inter- and intra- molecular disulfide bonds in chitinase, a cysteine-rich protein, cause aggregation when the recombinant protein is overexpressed in E. coli. Hence, aggregated proteins should be solubilized and allowed to refold to obtain native- or correctly- folded recombinant proteins.
  
  
  METHODS
  Dilution method that allows refolding of recombinant proteins, especially at high protein concentrations, is to slowly add the soluble protein to refolding buffer. For this purpose: first, the inclusion bodies containing insoluble proteins were purified; second, the aggregated proteins were solubilized; finally, the soluble proteins were refolded using glutathione redox system, guanidinium chloride, dithiothreitol, sucrose, and glycerol, simultaneously.
  
  
  RESULTS
  After protein solubilization and refolding, SDS-PAGE showed a 32 kDa band that was recognized by an anti-chitin antibody on western blots.
  
  
  CONCLUSIONS
  By this method, cysteine-rich proteins from E. coli inclusion bodies can be solubilized and correctly folded into active proteins.
authors.affiliation: Immunology Research Center, Medical School, Mashhad University of Medical Sciences, Mashhad, Iran.
authors.forename: Malihe
authors.initials: M
authors.lastname: Moghadam
authors.affiliation: Immunology Research Center, Medical School, Mashhad University of Medical Sciences, Mashhad, Iran.
authors.forename: Ali
authors.initials: A
authors.lastname: Ganji
authors.affiliation: Allergy Research Center, Medical School, Mashhad University of Medical Sciences, Mashhad, Iran.
authors.forename: Abdolreza
authors.initials: A
authors.lastname: Varasteh
authors.affiliation: Immunology Research Center, Iran University of Medical Sciences, Tehran, Iran; Department of Immunology, Iran University of Medical Sciences, Tehran, Iran.
authors.forename: Reza
authors.initials: R
authors.lastname: Falak
authors.affiliation: Immunology Research Center, Medical School, Mashhad University of Medical Sciences, Mashhad, Iran.
authors.forename: Mojtaba
authors.initials: M
authors.lastname: Sankian
keywords: Chitinase; Cysteine-rich proteins; Protein refolding; Protein solubilization