Overexpression in Escherichia coli, purification and characterization of the molecular chaperone HSC70.
The 70-kDa heat-shock cognate protein (HSC70), a constitutively expressed protein in mammalian cells, plays a major role in several cellular processes such as protein folding and assembly, uncoating of clathrin-coated vesicles and transport of protein through membranes. HSC70 has been overexpressed in Escherichia coli in a soluble form using a designed two-cistron expression vector, and purified to homogeneity in a two-step procedure involving ion-exchange and affinity chromatography. Up to 20 mg of pure protein could be obtained from 11 of cell culture. Amino-terminal sequencing of the recombinant protein gives the expected sequence, and non-denaturing gel electrophoresis as well as gel filtration analysis reveal the presence of self-associating species that could be dissociated by ATP. Crosslinking studies confirm the presence of multiple species and the dissociating effect of ATP. Temperatures above 42 degrees C induce the aggregation of HSC70; ATP shifts this effect to higher temperatures. The recombinant protein displays a low intrinsic ATPase activity that can be stimulated about threefold by binding to apocytochrome c, a permanently unfolded protein, while native cytochrome c has no effect on the ATPase activity indicating that recombinant HSC70 binds specifically unfolded protein but not their native counterpart. Thus, efficient production of recombinant HSC70 having structural and functional properties comparable to those of the natural protein could be achieved, thereby allowing the molecular basis of the chaperone function and its regulation through ATP hydrolysis to be probed.
Benaroudj
N
N
Laboratoire d'Enzymologie, CNRS, Gif-sur-Yvette, France.
Fang
B
B
Triniolles
F
F
Ghelis
C
C
Ladjimi
M M
MM
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000595:Amino Acid Sequence; D001483:Base Sequence; D001692:Biological Transport; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D002966:Clathrin; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D005822:Genetic Vectors; D050883:HSC70 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D006358:Hot Temperature; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D015183:Restriction Mapping; D017421:Sequence Analysis
D002352:Carrier Proteins; D002966:Clathrin; D050883:HSC70 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases