Isolation and characterization of human HSP70 expressed in Escherichia coli.
A plasmid containing the human HSP70 gene was used to transfect and express the protein in Escherichia coli. The bacterial product was a fusion protein containing 640 amino acids of HSP70, plus 33 additional NH2 terminal amino acids; 12 from the bacterial expression vector and 21 from a 5' human sequence that is not normally translated. It was partially purified by ion-exchange and ATP-Sepharose affinity column chromatography. The bacterially produced human HSP70 protein was then compared with HSP70 obtained from cultured 293 cells. Both shared the same staphylococcal V8 protease peptide fragment pattern, ATP binding, and a weak ATPase activity (about 10-15 nmol ATP hydrolyzed per milligram protein per minute at 30 degrees C). The bacterially produced human HSP70 protein differed in its V8 protease pattern with an E. coli ATP-binding protein that corresponded in molecular mass to the E. coli dnaK gene product. Mutants in the human HSP70 gene were constructed which significantly reduced a predicted major alpha-helical domain in the HSP70 molecule that has partial homology to an ATP-binding site of several protein kinases. One HSP70 mutant clone contained a deletion of 20% at the NH2 terminus, and expressed a 57-kDa product, while the other was missing the middle 50% of the gene (40-kDa product). Neither protein fragment bound to an ATP affinity column, suggesting that ATP binding to HSP70 may be conformationally affected by a region about 20% internal to the NH2 terminal end of the molecule. Recently, a similar location of the ATP-binding site has been reported by Milarski and Morimoto (27).
Macejak
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Department of Microbiology, Immunology and Parasitology, Louisiana State University Medical Center, New Orleans 70112-1393.
Rayfield
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Luftig
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D000251:Adenosine Triphosphatases / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D015153:Blotting, Western; D002460:Cell Line; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D005347:Fibroblasts / Q000378:metabolism; D015870:Gene Expression; D005796:Genes; D006360:Heat-Shock Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D006801:Humans; D007106:Immune Sera; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D012867:Skin / Q000378:metabolism
D006360:Heat-Shock Proteins; D007106:Immune Sera; D011993:Recombinant Fusion Proteins; D000251:Adenosine Triphosphatases