Cage” of the chaperonin, which has been estimated to be capable of housing proteins up 15900046 to 70 kDa in principle, with the actual size exclusion limit being somewhat less [36]. To ascertain whether MBP fusion proteins are capable of interacting productively with GroEL/S in vivo, we took advantageFigure 3. The effect of dnaJ, dnaK and tig gene deletions on the enzymatic activity of MBP-DHFR and MBP-G3PDH fusion proteins purified under native conditions. The data with error bars are expressed as mean 6 standard error of the mean (n = 3). The relative values were obtained by normalization with a standard 520-26-3 chemical information protein in each case. doi:10.1371/journal.pone.0049589.gThe Mechanism of Solubility Enhancement by MBPof a GroEL/S mutant (GroE3?) generated by directed evolution that is far more effective at stimulating the folding of GFP than is the wild-type 57773-63-4 chemical information chaperonin [22]. When GroE3? was co-expressed with the His6-MBP-GFP fusion protein (,70 kDa), the cells were significantly more fluorescent than they were when the wild-type chaperonin was co-expressed with the fusion protein or when only the fusion protein was overexpressed (Figure 4A). The increased fluorescence in the cells with GroE3? was a result of enhanced GFP folding because co-expression of GroE3? or wild-type GroE did not alter the amount of His6-MBP-GFP fusion protein that was produced (Figure 4B). Similar results were obtained when the even larger solubility enhancing tag NusA (,55 kDa) was joined to GFP to create an 82 kDa fusion protein (Figure S2).Interaction of Other Fusion Proteins with GroEL/S in E. coliIt was previously shown that a single amino acid substitution in MBP (I329W) dramatically decreases the solubility of several fusion proteins in E. 
coli but has no impact on the solubility of MBP in its unfused state [25]. The phenotype of this mutation was attributed to its effect on the equilibrium between the “open” and “closed” conformations of MBP, the latter being inhibitory to solubility enhancement. Intriguingly, we have found that the solubility defects of these fusion proteins can be rescued in whole or in part by co-expression of the GroEL/S chaperonin (Figure 6). Although the explanation for this effect remains to be elucidated, it constitutes further circumstantial evidence for an interaction between GroEL/S and MBP fusion proteins in E. coli. Moreover, the involvement of additional passenger proteins (e.g., human papilloma virus E6 and the tumor suppressor p16INK4a) suggests that the interaction of MBP fusion proteins with GroEL/S in vivo is not restricted to DHFR and G3PDH and may be a relatively common phenomenon.In vitro Refolding of MBP Fusions with GroEL/SSeeking to confirm that the GroEL/S chaperonin is involved in the folding of DHFR and G3PDH when these proteins are expressed as His6-MBP fusions in E. coli, we next performed in vitro refolding experiments in the presence of purified GroEL and ATP/Mg2+. The addition of GroEL alone did not improve the recovery of active passenger proteins in these cases (data not shown). However, the addition of GroES along with GroEL and ATP/Mg2+clearly stimulated the folding of both DHFR and G3PDH (Figure 5). These results are consistent with the hypothesis that GroEL/S plays an active role in the folding of the G3PDH and DHFR fusion proteins in E. coli.Discussion The Mechanism of Solubility Enhancement by MBPThe present study clearly demonstrates that the extraordinary ability of MBP to promote the solubility of its fusion.Cage” of the chaperonin, which has been estimated to be capable of housing proteins up 15900046 to 70 kDa in principle, with the actual size exclusion limit being somewhat less [36]. To ascertain whether MBP fusion proteins are capable of interacting productively with GroEL/S in vivo, we took advantageFigure 3. The effect of dnaJ, dnaK and tig gene deletions on the enzymatic activity of MBP-DHFR and MBP-G3PDH fusion proteins purified under native conditions. The data with error bars are expressed as mean 6 standard error of the mean (n = 3). The relative values were obtained by normalization with a standard protein in each case. doi:10.1371/journal.pone.0049589.gThe Mechanism of Solubility Enhancement by MBPof a GroEL/S mutant (GroE3?) generated by directed evolution that is far more effective at stimulating the folding of GFP than is the wild-type chaperonin [22]. When GroE3? was co-expressed with the His6-MBP-GFP fusion protein (,70 kDa), the cells were significantly more fluorescent than they were when the wild-type chaperonin was co-expressed with the fusion protein or when only the fusion protein was overexpressed (Figure 4A). The increased fluorescence in the cells with GroE3? was a result of enhanced GFP folding because co-expression of GroE3? or wild-type GroE did not alter the amount of His6-MBP-GFP fusion protein that was produced (Figure 4B). Similar results were obtained when the even larger solubility enhancing tag NusA (,55 kDa) was joined to GFP to create an 82 kDa fusion protein (Figure S2).Interaction of Other Fusion Proteins with GroEL/S in E. coliIt was previously shown that a single amino acid substitution in MBP (I329W) dramatically decreases the solubility of several fusion proteins in E. coli but has no impact on the solubility of MBP in its unfused state [25]. The phenotype of this mutation was attributed to its effect on the equilibrium between the “open” and “closed” conformations of MBP, the latter being inhibitory to solubility enhancement. Intriguingly, we have found that the solubility defects of these fusion proteins can be rescued in whole or in part by co-expression of the GroEL/S chaperonin (Figure 6). Although the explanation for this effect remains to be elucidated, it constitutes further circumstantial evidence for an interaction between GroEL/S and MBP fusion proteins in E. coli. Moreover, the involvement of additional passenger proteins (e.g., human papilloma virus E6 and the 
tumor suppressor p16INK4a) suggests that the interaction of MBP fusion proteins with GroEL/S in vivo is not restricted to DHFR and G3PDH and may be a relatively common phenomenon.In vitro Refolding of MBP Fusions with GroEL/SSeeking to confirm that the GroEL/S chaperonin is involved in the folding of DHFR and G3PDH when these proteins are expressed as His6-MBP fusions in E. coli, we next performed in vitro refolding experiments in the presence of purified GroEL and ATP/Mg2+. The addition of GroEL alone did not improve the recovery of active passenger proteins in these cases (data not shown). However, the addition of GroES along with GroEL and ATP/Mg2+clearly stimulated the folding of both DHFR and G3PDH (Figure 5). These results are consistent with the hypothesis that GroEL/S plays an active role in the folding of the G3PDH and DHFR fusion proteins in E. coli.Discussion The Mechanism of Solubility Enhancement by MBPThe present study clearly demonstrates that the extraordinary ability of MBP to promote the solubility of its fusion.
