Kinetic and structural characterization of human mortalin.
Human mortalin is an Hsp70 chaperone that has been implicated in cancer, Alzheimer's and Parkinson's disease, and involvement has been suggested in cellular iron-sulfur cluster biosynthesis. However, study of this important human chaperone has been hampered by a lack of active material sufficient for biochemical characterization. Herein, we report the successful purification and characterization of recombinant human mortalin in Escherichia coli. The recombinant protein was expressed in the form of inclusion bodies and purified by Ni-NTA affinity chromatography. The subsequently refolded protein was confirmed to be active by its ATPase activity, a characteristic blue-shift in the fluorescence emission maximum following the addition of ATP, and its ability to bind to a likely physiological substrate. Single turnover kinetic experiments of mortalin were performed and compared with another Hsp70 chaperone, Thermotogamaritima DnaK; with each exhibiting slow ATP turnover rates. Secondary structures for both chaperones were similar by circular dichroism criteria. This work describes an approach to functional expression of human mortalin that provides sufficient material for detailed structure-function studies of this important Hsp70 chaperone.
Ohio State Biochemistry Program, The Ohio State University, Columbus, OH 43210, United States.
Wen-I
WI
Luo
Eric
E
Dizin
Taejin
T
Yoon
James A
JA
Cowan
D000255:Adenosine Triphosphate / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D002846:Chromatography, Affinity; D004926:Escherichia coli / Q000235:genetics*; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D018832:Molecular Chaperones / Q000737:chemistry / Q000378:metabolism; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity
D001426:Bacterial Proteins; D018840:HSP70 Heat-Shock Proteins; D018832:Molecular Chaperones; D011994:Recombinant Proteins; C080352:mortalin; D000255:Adenosine Triphosphate