Characterization of interactions between Escherichia coli molecular chaperones and immobilized caseins.
The molecular chaperones were affinity purified with immobilized alpha-casein (45mg protein/g beads) and beta-casein columns (30 mg protein/g beads) from two heat-induced E. coli strains, NM522 and BL21. After removing nonspecifically bound proteins with 1 M NaCl, the molecular chaperones were eluted with cold water, 1 mM Mg-ATP, or 6 M urea. The eluates from affinity columns were analyzed by SDS-PAGE and Western analysis. Western analysis identified five E. coli molecular chaperones including DnaK, DnaJ, GrpE, GroEL, and GroES in eluates. Among samples, ATP eluates showed the highest chaperone purity of 80-87% followed by cold water eluates with 62-68% purity. The beta-casein column showed a higher chaperone binding capacity than the alpha-casein column. A higher concentration of chaperones was purified from strain BL21 than strain NM522 which may have been due to the lack of lon protease in the BL21 strain.
Nam
Seung-Hee
SH
Department of Nutrition and Food Sciences, Utah State University, Logan, Utah 84322-8700, USA.
Walsh
Marie K
MK
D000255:Adenosine Triphosphate; D024401:Carbonic Anhydrase I; D002364:Caseins / Q000737:chemistry*; D002846:Chromatography, Affinity / Q000379:methods; D003080:Cold Temperature; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018832:Molecular Chaperones / Q000032:analysis / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D008970:Molecular Weight; D020033:Protein Isoforms / Q000737:chemistry; D014508:Urea
D002364:Caseins; D018832:Molecular Chaperones; D020033:Protein Isoforms; D000255:Adenosine Triphosphate; D014508:Urea; D024401:Carbonic Anhydrase I