CHAPERONE COEXPRESSION PLASMIDS - DIFFERENTIAL AND SYNERGISTIC ROLES OF DNAK-DNAJ-GRPE AND GROEL-GROES IN ASSISTING FOLDING OF AN ALLERGEN OF JAPANESE CEDAR POLLEN, CRYJ2 IN ESCHERICHIA-COLI

Plasmids that can be used for controlled expression of the DnaK-DnaJ-G rpE and/or GroEL-GroES chaperone team were constructed in order to fac ilitate assessment of the effects of these chaperone teams on folding or assembly of recombinant proteins in Escherichia call. A typical pAC YC183-based plasmid which was obtained could express the major DnaK-Dn aJ-GrpE and GroEL-GroES chaperone teams from separate promoters when L -arabinose and tetracycline, respectively, were added in a dose-depend ent fashion. The model protein used to determine whether this system w as useful was an allergen of Japanese cedar pollen, Cryj2, which was u nstable when it was produced in E. coli K-12. The effects of chaperone coexpression on the folding, aggregation, and stability of Cryj2 were examined in the wild type and in several mutant bacteria. Coexpressio n of the DnaK-DnaJ-GrpE and/or GroEL-GroES chaperone team at appropria te levels resulted in marked stabilization and accumulation of Cryj2 w ithout extensive aggregation. Experiments performed with mutants that lack each of the chaperone proteins (DnaK, DnaJ, GrpE, GroEL, and GroE S) or heat shock transcription factor sigma(32) revealed that both cha perone teams are critically involved in Cryj2 folding but that they ar e involved in distinct ways. In addition, it was observed that the two chaperone teams have synergistic roles in preventing aggregation of C ryj2 in the absence of sigma(32) at certain temperatures.