Biotechnology and Bioprocess Engineering:BBE

The Korean Society for Biotechnology and Bioengineering (한국생물공학회)
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Isolation of Proteins that Specifically Interact with the ATPase Domain of Mammalian ER Chaperone, BiP

  • Chung, Kyung-Tae (Department of Applied Life Science, College of Natural Sciences, Dong-Eui University) ;
  • Lee, Tae-Ho (Department of Microbiology, College of Natural Sciences, Pusan National University) ;
  • Kang, Gyong-Suk (Department of Pharmaceutical, University of Tennessee, The Health Science Center, Memphis)
  • Published : 2003.06.01
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Abstract

BiP, immunoglobulin binding protein, is an ER homologue of Hsp70. However, unlit other Hsp70 proteins, regulatory protein(s) for BiP has not been identified. Here, we demo strafed the presence of potential regulatory proteins for BiP using a pull -down assay. Since BiP can bind any unfolded protein, only the ATPase domain of BiP was used for the pull -down assay in order to minimize nonspecific binding. The ATPase domain was cloned to produce recombinant protein, which was then conjugated to CNBr-activated agarose. The structural conformation and ATP hydrolysis activity of the recombinant ATPase domain were similar to those of the native protein, light proteins from metabolically labeled mouse plasmacytoma cells specifically bound to the recombinant ATPase protein. The binding of these proteins was inhibited by excess amounts of free ATPase protein, and was dependent on the presence of ATP. These proteins were eluted by ADP. Of these proteins, Grp170 and BiP where identified. while the other were not identified as known ER proteins, from Western blot analyses. The presence of the ATPase-binding proteins for BiP was first demonstrated in this study, and our data suggest similar regulatory machinery for BiP may exist in the ER, as found in prokaryotes and other cellular compartments.

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References

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