High-level expression of soluble rat hsc70 in Escherichia coli: purification and characterization of the cloned enzyme.
We have cloned the cDNA of rat hsc70 (clathrin-uncoating ATPase) into a T7 expression system and have expressed this enzyme in Escherichia coli. The recombinant clathrin-uncoating ATPase is in the cytosolic fraction of the bacterium and is soluble. It was purified to homogeneity by DEAE-cellulose and ATP-agarose column chromatography. From 1 litre of bacterial culture (0.3-0.4 g of proteins), 5-20 mg of pure recombinant clathrin-uncoating ATPase was routinely obtained. The cloned enzyme is capable of dissociating clathrin from bovine coated vesicle. Furthermore, it is not methylated on basic amino acid residues and is not blocked at the N-terminus, indicating that these modifications on hsc70 are not essential for uncoating of clathrin. Binding of [alpha-32P]ATP by purified recombinant hsc70 was analysed by Scatchard plot. The results indicate that there one high-affinity binding component with a Kd (dissociation constant) of 0.2-0.3 microM. The peptide-stimulated ATPase activities of recombinant hsc70 at 37 degrees C with respect to S-peptide peptides P3a and GT4 at a concentration of 1.2 mM are 142 +/- 6, 214 +/- 8 and 362 +/- 5 pmol/h per micrograms of hsc70 protein respectively. The EC50 values of hsc70 ATPase for S-peptide, peptides P3a and GT4 are 2, 0.67 and 0.17 mM respectively. On the other hand, the dissociation constants of S-peptide, peptides P3a and GT4 for recombinant hsc70 are 7.6, 13 and 100 microM respectively. Thus peptide GT4 is the only peptide examined for which the binding constant is comparable with the EC50 for stimulation ATPase activity, albeit it has the lowest affinity for hsc70.
Wang
C
C
Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan.
Lee
M R
MR
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001345:Autoradiography; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D003001:Cloning, Molecular; D004247:DNA; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D050883:HSC70 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D008969:Molecular Sequence Data; D010455:Peptides / Q000378:metabolism; D010957:Plasmids; D051381:Rats; D013379:Substrate Specificity
D002352:Carrier Proteins; D050883:HSC70 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; C494373:Hspa8 protein, rat; D010455:Peptides; D000255:Adenosine Triphosphate; D004247:DNA; D000251:Adenosine Triphosphatases