Isolation, expression, and characterization of fully functional nontoxic BiP/GRP78 mutants

Mammalian BiP/GRP78 and Escherichia coli DnaK belong to the highly conserve d hsp70 family and function as molecular chaperones in the endoplasmic reti culum or the cytosol, respectively. Induction of murine BiP/GRP78 expressio n in E. coli leads to growth arrest and cell death, independent of the bact erial strain and vector used. Analysis of various BiP constructs and mutant s shows that the dominant-lethal phenotype is induced specifically by the e xpression of the 13.7-kDa C-terminal domain and abolished by a single subst itution in that region, Deletion of that region also results in nontoxic ge ne products that can be overexpressed and purified to homogeneity. The nont oxic mutants are highly expressed in E. coli, representing up to 20% of the soluble fraction. They are catalytically active, depolymerize upon binding ATP or synthetic peptide, and interact with the J-domain of the DnaJ-like accessory protein, MTJ1, with near wild-type affinity. Our data indicate th at the cytotoxic effect encountered during overexpression of recombinant pr oteins can be caused by a single domain and can be alleviated by a specific mutation or deletion in that region without altering the catalytic propert ies of the enzyme. (C) 2001 Academic Press.