Date of Award:

5-2002

Document Type:

Dissertation

Degree Name:

Doctor of Philosophy (PhD)

Department:

Nutrition, Dietetics, and Food Sciences

Department name when degree awarded

Nutrition and Food Sciences

Committee Chair(s)

Marie K. Walsh

Committee

Marie K. Walsh

Committee

Donald McMahon

Committee

Daren Cornforth

Committee

Lance Seefeldt

Committee

Timothy Taylor

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.

Heat-induced Escherichia coli BL21 cell lysate (10 mg protein) was applied to immobilized ɑ-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 ɑ-casein column since β-casein urea eluates contained 3.18 mg total protein (or 58% chaperone) compared to a-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.

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