title: Affinity purification and characterization of the Escherichia coli molecular chaperones.
abstract: The molecular chaperones are a group of proteins that are effective in vitro and in vivo folding aids and show a well-documented affinity for proteins lacking tertiary structure. The molecular chaperones were induced from lon(-) Escherichia coli mutants, affinity purified with an immobilized beta-casein column, and assayed for refolding activity with thermally and chemically denatured carbonic anhydrase B (CAB). Chaperones were induced with three treatments: heat shock at 39 degrees C, heat shock 42 degrees C, and alcohol shock with 3% ethanol (v/v). Lysates were applied to an immobilized beta-casein (30 mg/g beads) column. After removing nonspecifically bound proteins with 1 M NaCl, the molecular chaperones were eluted with cold water or 1 mM Mg-ATP. The cold water and Mg-ATP eluates were analyzed by SDS-PAGE. Western analysis identified five E. coli molecular chaperones including DnaK, DnaJ, GrpE, GroEL, and GroES. The purity of eluted chaperones was 58% with cold water and 100% with Mg-ATP. Refolding denatured CAB in the presence of Mg-ATP resulted in a 97% recovery of heat-denatured CAB and a 68% recovery of chemically denatured CAB. The use of affinity matrices for the chaperone purification which are effective as in vitro folding aids will be presented.
authors.affiliation: Department of Nutrition and Food Sciences, Utah State University, Logan, Utah 84322-8700, USA.
authors.forename: Seung-Hee
authors.initials: SH
authors.lastname: Nam
authors.forename: Marie K
authors.initials: MK
authors.lastname: Walsh
mesh_terms: D001426:Bacterial Proteins / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification*; D015153:Blotting, Western; D002364:Caseins; D002846:Chromatography, Affinity; D004926:Escherichia coli / Q000737:chemistry*; D018832:Molecular Chaperones / Q000032:analysis / Q000302:isolation & purification*; D017510:Protein Folding; D020673:Protein Renaturation
chemical_list: D001426:Bacterial Proteins; D002364:Caseins; D018832:Molecular Chaperones