The grpE protein of Escherichia coli. Purification and properties.
The grpE gene of Escherichia coli was first identified because a mutation in it, grpE280, prevented bacteriophage lambda DNA replication in vivo. Subsequent work resulted in the identification of the grpE protein in two-dimensional gels and its classification as a heat shock protein. Here we report the purification of the grpE protein. We show that overproduction of grpE occurs in dnaK 103 bacteria which do not produce a functional Mr 72,000 dnaK protein. The grpE protein was purified from this strain primarily by its specific retention on a dnaK affinity column. The interaction between these two proteins, which is stable in the presence of 2 M KCl, allowed other proteins to be washed from this column. grpE was then eluted by ATP, which disrupts the interaction. During purification, grpE activity was monitored by its ability to complement an in vitro lambda dv DNA replication system dependent on the lambda O and lambda P proteins. The effect of ATP on the dnaK-grpE complex was also observed during sedimentation of the two proteins in glycerol gradients. Purified grpE protein has a Mr of approximately 23,000 under both denaturing and native conditions, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and sedimentation, respectively. However, in the presence of dnaK under native conditions, grpE cosediments with dnaK. When ATP is added to the gradient, the complex is disrupted, and the two proteins sediment independently as monomers.
Zylicz
M
M
Department of Cellular, Viral, and Molecular Biology, University of Utah, Salt Lake City 84132.
Ang
D
D
Georgopoulos
C
C
D000255:Adenosine Triphosphate / Q000494:pharmacology; D001426:Bacterial Proteins / Q000302:isolation & purification*; D002846:Chromatography, Affinity; D004261:DNA Replication; D004926:Escherichia coli / Q000032:analysis*; D008970:Molecular Weight; D010957:Plasmids; D011489:Protein Denaturation
D001426:Bacterial Proteins; D000255:Adenosine Triphosphate