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-GrpE and/or GroEL-GroES chaperone team were constructed in order to facilitate assessment of the effects of these chaperone teams on folding or assembly or recombinant proteins in Escherichia coli. A typical pACYC184-based plasmid which was obtained could express the major DnaK-DnaJ-GrpE and GroEL-GroES chaperone teams from separate promoters when L-arabinose and tetracycline, respectively, were added in a dose-dependent fashion. The model protein used to determine whether this system was useful was an allergen of Japanese cedar pollen, Cryj2, which was unstable 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. Coexpression of the DnaK-DnaJ-GrpE and/or GroEL-GroES chaperone team at appropriate levels resulted in marked stabilization and accumulation of Cryj2 without extensive aggregation. Experiments performed with mutants that lack each of the chaperone proteins (DnaK, DnaJ, GrpE, GroEL, and GroES) or heat shock transcription factor sigma 32 revealed that both chaperone teams are critically involved in Cryj2 folding but that they are involved in distinct ways. In addition, it was observed that the two chaperone teams have synergistic roles in preventing aggregation of Cryj2 in the absence of sigma 32 at certain temperatures.
Nishihara
K
K
HSP Research Institute, Kyoto Research Park, Japan.
Kanemori
M
M
Kitagawa
M
M
Yanagi
H
H
Yura
T
T
D000485:Allergens / Q000096:biosynthesis*; D001426:Bacterial Proteins / Q000502:physiology*; D018835:Chaperonin 10 / Q000502:physiology*; D018834:Chaperonin 60 / Q000502:physiology*; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins; D050956:HSP40 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins / Q000502:physiology*; D006360:Heat-Shock Proteins / Q000502:physiology*; D010957:Plasmids; D011058:Pollen / Q000276:immunology*; D017510:Protein Folding; D011994:Recombinant Proteins / Q000096:biosynthesis*
D000485:Allergens; D001426:Bacterial Proteins; D018835:Chaperonin 10; D018834:Chaperonin 60; C494635:DnaJ protein, E coli; D029968:Escherichia coli Proteins; C068113:GrpE protein, Bacteria; C489400:GrpE protein, E coli; D050956:HSP40 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D011994:Recombinant Proteins; C063842:dnaK protein, E coli