| 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.; Heat-induced Escherichia coli BL21 cell lysate (10 mg protein) was applied to immobilized α-casein (45 mg/g beads) or β-casein (30 mg/g beads) column. After removing a majority of nonspecifically bound proteins with 1 M NaCl, the molecular chaperones were eluted with cold water, 1 mM Mg-ATP, or 6 M urea. Western analysis identified five Escherichia coli molecular chaperones including DnaK, DnaJ, GrpE, GroEL, and GroES. Among samples, ATP eluates showed the highest chaperone purity of 80–87% followed by cold water eluates with 62–68% purity. The β-Casein column showed a higher binding capacity than the α-casein column since β-casein urea eluates contained 3.18 mg total protein (or 58% chaperone) compared to α-casein urea eluates with 2.68 mg total protein (or 32% chaperone). For strain comparison, Escherichia coli NM522 eluates showed more unidentified proteins in cold water eluates from both affinity columns.; Chaperones were induced from BL21 strain with three treatments: heat shock at 39°C, heat shock at 42°C, and alcohol shock with 3% ethanol (v/v). Lysates were applied to an immobilized β-casein (30 mg/g beads) column. The molecular chaperones were eluted with cold water or 1 mM Mg-ATP after washing with 1 M NaCl. The purity of eluted chaperones was 58% with cold water and 100% with Mg-ATP. The treatment at 42°C was the most efficient for chaperone induction with highest chaperone yield of 1.0 mg among samples. Refolding denatured carbonic anhydrase B enzyme in the presence of Mg-ATP resulted in a 97% recovery of heat-denatured enzyme and a 68% recovery of chemically denatured enzyme.; It was concluded that the novel casein affinity chromatography is a rapid and efficient method for purification of chaperone. The affinity purified chaperones were effective in vitro folding aids. |
