title: Folding machineries displayed on a cation-exchanger for the concerted refolding of cysteine- or proline-rich proteins.
abstract: BACKGROUND
  Escherichia coli has been most widely used for the production of valuable recombinant proteins. However, over-production of heterologous proteins in E. coli frequently leads to their misfolding and aggregation yielding inclusion bodies. Previous attempts to refold the inclusion bodies into bioactive forms usually result in poor recovery and account for the major cost in industrial production of desired proteins from recombinant E. coli. Here, we describe the successful use of the immobilized folding machineries for in vitro refolding with the examples of high yield refolding of a ribonuclease A (RNase A) and cyclohexanone monooxygenase (CHMO).
  
  
  RESULTS
  We have generated refolding-facilitating media immobilized with three folding machineries, mini-chaperone (a monomeric apical domain consisting of residues 191-345 of GroEL) and two foldases (DsbA and human peptidyl-prolyl cis-trans isomerase) by mimicking oxidative refolding chromatography. For efficient and simple purification and immobilization simultaneously, folding machineries were fused with the positively-charged consecutive 10-arginine tag at their C-terminal. The immobilized folding machineries were fully functional when assayed in a batch mode. When the refolding-facilitating matrices were applied to the refolding of denatured and reduced RNase A and CHMO, both of which contain many cysteine and proline residues, RNase A and CHMO were recovered in 73% and 53% yield of soluble protein with full enzyme activity, respectively.
  
  
  CONCLUSIONS
  The refolding-facilitating media presented here could be a cost-efficient platform and should be applicable to refold a wide range of E. coli inclusion bodies in high yield with biological function.
authors.affiliation: Department of Agricultural Biotechnology, Seoul National University, Seoul 151-921, Korea. dlee@bioeng.ucsd.edu
authors.forename: Dae-Hee
authors.initials: DH
authors.lastname: Lee
authors.forename: Sung-Gun
authors.initials: SG
authors.lastname: Kim
authors.forename: Dae-Hyuk
authors.initials: DH
authors.lastname: Kweon
authors.forename: Jin-Ho
authors.initials: JH
authors.lastname: Seo