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Isolation, expression, and characterization of fully functional nontoxic BiP/GRP78 mutants
L S King1, M Berg, M Chevalier
1Center for Pharmaceutical Biotechnology, Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago, College of Pharmacy, Molecular Biology Research Building, 900 South Ashland Avenue, Chicago, IL 60607, USA.
Protein Expression and Purification
|June 5, 2001
Summary
Expressing mammalian BiP/GRP78 in E. coli causes bacterial death, but a specific C-terminal domain is responsible. Modifying this domain eliminates toxicity without affecting chaperone function.
Area of Science:
- Molecular Biology
- Biochemistry
- Microbiology
Background:
- Mammalian BiP/GRP78 and E. coli DnaK are hsp70 molecular chaperones.
- BiP functions in the endoplasmic reticulum, DnaK in the cytosol.
Purpose of the Study:
- Investigate the cause of growth arrest and cell death in E. coli expressing murine BiP/GRP78.
- Identify specific domains or mutations responsible for the toxic effects.
Main Methods:
- Expression of various BiP/GRP78 constructs and mutants in E. coli.
- Analysis of bacterial growth, cell viability, and protein expression levels.
- Biochemical assays to assess chaperone activity and protein interactions.
Main Results:
- Murine BiP/GRP78 expression induced dominant lethality in E. coli.
- The 13.7-kDa C-terminal domain was identified as the sole inducer of the toxic phenotype.
- A single substitution or deletion in this domain abolished toxicity, allowing high-level expression of active, non-toxic BiP.
Conclusions:
- The cytotoxic effect of BiP/GRP78 overexpression in E. coli is domain-specific.
- Targeted mutations or deletions can alleviate toxicity without compromising catalytic activity.
- This finding is crucial for the successful overexpression and purification of recombinant BiP/GRP78.