title: Folding and purification of insoluble (inclusion body) proteins from Escherichia coli.
abstract: Heterologous expression of recombinant proteins in E. coli often results in the formation of insoluble and inactive protein aggregates, commonly referred to as inclusion bodies. To obtain the native (i.e., correctly folded) and hence active form of the protein from such aggregates, four steps are usually followed: (1) the cells are lysed and the are aggregates, (2) the cell wall and outer membrane components of the aggregates are removed, (3) the aggregates are solubilized (or extracted) with strong protein denaturants, and (4) the solubilized, denatured proteins are folded with concomitant oxidation of reduced cysteine residues into the correct disulfide bonds to obtain the native protein. This unit features three different approaches to the final step of protein folding and purification. In the first, guanidine HCl is used as the denaturant, after which the solubilized protein is folded (before purification) in an "oxido-shuffling" buffer system to increase the rate of protein oxidation. In the second, acetic acid is used to solubilize the protein which is then partially purified by gel filtration before folding, and then the protein is folded and oxidized by simple dialyzed against water. A Support Protocol is included for rapidly determining the amount of folded protein that contains the correct disulfide linkage pattern. Finally, folding and purification of a fusion protein is described using metal-chelate affinity chromatography.
authors.affiliation: National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: P T
authors.initials: PT
authors.lastname: Wingfield
authors.forename: I
authors.initials: I
authors.lastname: Palmer
authors.forename: S M
authors.initials: SM
authors.lastname: Liang
mesh_terms: D002846:Chromatography, Affinity; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D002479:Inclusion Bodies / Q000737:chemistry* / Q000378:metabolism; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins