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

Abstract

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.

FIG. 1

Structure of chaperone coexpression plasmid pG-KJE6. ori, replication origin of pACYC184; cat, chloramphenicol acetyltransferase gene; Pzt-1p, Pzt-1 promoter; tetR, tetR repressor gene; araBp/o, araB promoter-operator; araC, araC repressor gene.

FIG. 2

Expression and stability of Cryj2 in the wild type and in isogenic chaperone mutants harboring pKCJ2l. (A) Level of expression and fractionation of Cryj2. Cells were grown to the mid-log phase in L broth at 30°C, and Cryj2 was induced with IPTG (1 mM) for 2 h and analyzed by immunoblotting with whole-cell proteins (Total) or with the soluble (lanes S) or insoluble (lanes I) fractions obtained after centrifugation (Fractions), as described in the text. The arrowhead indicates the position of Cryj2. (B) Stability of Cryj2 in vivo. The fraction of Cryj2 remaining after incubation in the presence of spectinomycin (500 μg/ml) was plotted versus incubation time. Averages of data from three experiments are shown. Symbols: ○, MC4100 (wild type); •, ΔdnaK52 mutant; ▵, dnaJ259 mutant; ▴, grpE280 mutant; □, groEL44 mutant; ▪, groES72 mutant.

FIG. 3

Expression and stability of Cryj2 in the wild type and in the isogenic ΔrpoH mutant harboring pKCJ2l. (A) Cells were grown in L broth at 20°C, Cryj2 was induced for 3 h, and cells were analyzed by immunoblotting before (left) and after (right) soluble (lanes S) and insoluble (lanes I) fractions were separated, essentially as described in the legend to Fig. 2. (B) Stability of Cryj2 in vivo, determined as described in the legend to Fig. 2. Averages of data from three experiments are shown. Symbols: ○, MG1655 (wild type); •, NK161 (ΔrpoH).

FIG. 4

Stabilization of Cryj2 by coexpression of the DnaK-DnaJ-GrpE chaperone team in the wild-type strain. Strain MG1655 harboring pKCJ2l and pG-KJE6 was grown in L broth containing various concentrations of l-arabinose (Ara) at 30°C for three or four generations, and Cryj2 was induced with IPTG (1 mM) for 2 h. (A) Expression of the DnaK-DnaJ-GrpE chaperone team as determined by SDS-PAGE followed by staining with Coomassie brilliant blue. l-Arabinose was added to lanes 1 through 5 as indicated. (B and C) Level of expression and fractionation of Cryj2 (B) and stability of Cryj2 in vivo (C) determined essentially as described in the legend to Fig. 2. Symbols: ○, no arabinose (control); •, 4 mg of l-arabinose per ml (chaperone coexpression). Lanes S, soluble fractions; lanes I, insoluble fractions.

FIG. 5

Stabilization of Cryj2 by coexpression of the GroEL-GroES chaperone team in the wild-type strain. Strain MG1655 harboring pKCJ2l and pG-KJE6 was grown in L broth containing various concentrations of tetracycline (Tet) at 30°C, and Cryj2 was induced with IPTG. The GroE chaperones induced with tetracycline were monitored (A) and the level of expression and fractionation of Cryj2 (B) and the stability of Cryj2 in vivo (C) were determined essentially as described in the legends to Fig. 2 and 4. Overexpression of GroES was confirmed by SDS-PAGE and immunoblotting (data not shown). Symbols: ○, no tetracycline (control); •, 10 ng of tetracycline per ml (chaperone coexpression). Lanes S, soluble fractions; lanes I, insoluble fractions.

FIG. 6

Effect of chaperone coexpression on disaggregating Cryj2 in the ΔrpoH mutant at 20°C. A pair of NK161 (ΔrpoH) strains harboring pKCJ2l and pKJE7 or pGro7 were grown at 20°C, each chaperone team was induced with arabinose (Ara), and Cryj2 was induced with IPTG for 3 h. The levels of expression of chaperones (A) and the levels of expression of Cryj2 before (B) and after (C) fractionation were determined essentially as described in the legends to Fig. 2 and 4. Lanes S, soluble fractions; lanes I, insoluble fractions.

FIG. 7

Synergistic effect of chaperones on preventing Cryj2 aggregation in the ΔrpoH mutant at 30°C. Strain NK161 (ΔrpoH) harboring pKCJ2l and pG-KJE6 was grown at 30°C, either or both chaperone teams were induced, and Cryj2 was induced for 2 h. The levels of chaperones expressed (A) and the levels of Cryj2 before (B) and after (C) fractionation were determined as described in the legends to Fig. 2 and 4. Lanes S, soluble fractions; lanes I, insoluble fractions. Ara, arabinose; Tet, tetracycline.