PMID:11858724
| Citation |
Nam, SH and Walsh, MK (2002) Affinity purification and characterization of the Escherichia coli molecular chaperones. Protein Expr. Purif. 24:282-91 |
|---|---|
| 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. |
| Links |
PubMed Online version:10.1006/prep.2001.1571 |
| Keywords |
Bacterial Proteins/analysis; Bacterial Proteins/chemistry; Bacterial Proteins/isolation & purification; Blotting, Western; Caseins; Chromatography, Affinity; Escherichia coli/chemistry; Molecular Chaperones/analysis; Molecular Chaperones/isolation & purification; Protein Folding; Protein Renaturation |
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