Human stress protein hsp70: overexpression in E coli, purification and characterization.
The gene encoding the stress-inducible member of human heat shock protein hsp70, was expressed in E. coli using the bacteriophage T7 RNA polymerase-based gene expression system. Recombinant hsp70 (R-hsp70) was purified from inclusion bodies after solubilization and refolding, using a combination of ATP-agarose affinity chromatography and ion-exchange chromatography. R-hsp70 was shown to be monomeric and free of its structurally similar E. coli counterpart, DnaK. In addition, R-hsp70 is functional as demonstrated by its ability to bind to peptides and to ATP. The availability of pure, correctly folded R-hsp70 in sufficient quantity will assist in the structural and functional characterization of hsp70. Furthermore, an understanding of the cytoprotective function of hsp70 and its role in immune responses during infections will be facilitated by the availability of pure R-hsp70.
Jindal
S
S
PerSeptive Biosystems, Framingham, MA, USA.
Murray
P
P
Rosenberg
S
S
Young
R A
RA
Williams
K P
KP
D000255:Adenosine Triphosphate / Q000378:metabolism; D001667:Binding, Competitive; D002458:Cell Fractionation; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D012321:DNA-Directed RNA Polymerases; D004926:Escherichia coli / Q000235:genetics* / Q000648:ultrastructure; D015870:Gene Expression; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D002479:Inclusion Bodies / Q000737:chemistry; D013058:Mass Spectrometry; D010455:Peptides / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D014764:Viral Proteins
D018840:HSP70 Heat-Shock Proteins; D010455:Peptides; D011994:Recombinant Proteins; D014764:Viral Proteins; D000255:Adenosine Triphosphate; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases