Nd folded) fusion partner would be expected to exert a similar entropic effect on the folding of the attached protein and promote its solubility, which is not the case. Neither the micelle nor the entropic-anchor model can readily account for the observation that only a subset of highly soluble proteins, such as MBP, are effective solubilizing agents. Yet another theory is that solubility-enhancing fusion partners act as “chaperone magnets” and solubility results from interactions with endogenous chaperones [9]. Finally, it has been proposed thatThe Mechanism of Solubility Enhancement by MBPsolubility enhancers may have an innate, passive chaperone-like quality that I-BRD9 chemical information Cyproconazole manifests itself as iterative cycles of transient intramolecular binding to passenger proteins in a manner that prevents their self-association and aggregation [4,10,11,12]. In an effort to illuminate the mechanism by which MBP, a universally acknowledged solubility-enhancing tag [13,14,15,16,17], promotes the solubility of its fusion partners, we have conducted refolding experiments with MBP fusion proteins in vitro. Additionally, we have examined how passenger proteins fold when fused to MBP, both in vitro and in vivo. Our results indicate that MBP has an intrinsic ability to solubilize its fusion partners that does not depend on any exogenous factors. Further, we present evidence that there are at least two pathways to the native state: passenger proteins either fold spontaneously or they are assisted by endogenous chaperones in vivo.Materials and Methods Construction of Expression VectorsVarious protein expression vectors were constructed by 1480666 Gateway cloning (Life Technologies Inc., Carlsbad, CA), using the destination vectors pDEST-527, pDEST-565 (Protein Expression Laboratory, SAIC-Frederick, Frederick, MD, USA) and pDEST-HisMBP [18]. The standard LR reaction was employed throughout as per the manufacturer’s protocol. A two-step PCR procedure was used to construct Gateway entry clones of the passenger proteins. The open reading frames or entry clones encoding green fluorescent protein (GFP) [4], glyceraldehyde 3phosphate dehydrogenase (G3PDH) [6], dihydrofolate reductase (DHFR) [6], dual 24272870 specificity phosphatase 14 (DUSP14) [19], and tobacco etch virus (TEV) protease [20] were described previously. In each case, a pair of gene-specific primers was utilized in a PCR reaction with the appropriate plasmid template and then the PCR amplicon from this reaction was used as the template for a second round of PCR with the forward primer PE-277 (59-GGGG ACA AGT TTG TAC AAA AAA GCA GGC TCG GAG AAC CTG TAC TTC CAG-39) and the gene-specific reverse primer (Table 1). The final PCR amplicons were recombined into pDONR221 (Life Technologies) to generate the entry clones, except for GFP and G3PDH, which were recombined into pDONR201 (Life Technologies) instead. All of the entry clones were subsequently recombined in LR reactions with the destination vectors mentioned above. The resulting protein expression vectors encoded either His6- (pDEST-527 in the LR reaction), His6-GST (pDEST-565 in the LR reaction), or His6-MBP (pDEST-HisMBP in the LR reaction) tags appended to the Ntermini of the passenger proteins along with canonical TEV protease recognition sites (ENLYFQG) between the tags and the passengers (except for the vectors encoding TEV protease fusions, which contained the uncleavable recognition site ENLYFQP [21] instead). The pDEST-HisMBP derivative carrying an I329W mutation in.Nd folded) fusion partner would be expected to exert a similar entropic effect on the folding of the attached protein and promote its solubility, which is not the case. Neither the micelle nor the entropic-anchor model can readily account for the observation that only a subset of highly soluble proteins, such as MBP, are effective solubilizing agents. Yet another theory is that solubility-enhancing fusion partners act as “chaperone magnets” and solubility results from interactions with endogenous chaperones [9]. Finally, it has been proposed thatThe Mechanism of Solubility Enhancement by MBPsolubility enhancers may have an innate, passive chaperone-like quality that manifests itself as iterative cycles of transient intramolecular binding to passenger proteins in a manner that prevents their self-association and aggregation [4,10,11,12]. In an effort to illuminate the mechanism by which MBP, a universally acknowledged solubility-enhancing tag [13,14,15,16,17], promotes the solubility of its fusion partners, we have conducted refolding experiments with MBP fusion proteins in vitro. Additionally, we have examined how passenger proteins fold when fused to MBP, both in vitro and in vivo. Our results indicate that MBP has an intrinsic ability to solubilize its fusion partners that does not depend on any exogenous factors. Further, we present evidence that there are at least two pathways to the native state: passenger proteins either fold spontaneously or they are assisted by endogenous chaperones in vivo.Materials and Methods Construction of Expression VectorsVarious protein expression vectors were constructed by 1480666 Gateway cloning (Life Technologies Inc., Carlsbad, CA), using the destination vectors pDEST-527, pDEST-565 (Protein Expression Laboratory, SAIC-Frederick, Frederick, MD, USA) and pDEST-HisMBP [18]. The standard LR reaction was employed throughout as per the manufacturer’s protocol. A two-step PCR procedure was used to construct Gateway entry clones of the passenger proteins. The open reading frames or entry clones encoding green fluorescent protein (GFP) [4], glyceraldehyde 3phosphate dehydrogenase (G3PDH) [6], dihydrofolate reductase (DHFR) [6], dual 24272870 specificity phosphatase 14 (DUSP14) [19], and tobacco etch virus (TEV) protease [20] were described previously. In each case, a pair of gene-specific primers was utilized in a PCR reaction with the appropriate plasmid template and then the PCR amplicon from this reaction was used as the template for a second round of PCR with the forward primer PE-277 (59-GGGG ACA AGT TTG TAC AAA AAA GCA GGC TCG GAG AAC CTG TAC TTC CAG-39) and the gene-specific reverse primer (Table 1). The final PCR amplicons were recombined into pDONR221 (Life Technologies) to generate the entry clones, except for GFP and G3PDH, which were recombined into pDONR201 (Life Technologies) instead. All of the entry clones were subsequently recombined in LR reactions with the destination vectors mentioned above. The resulting protein expression vectors encoded either His6- (pDEST-527 in the LR reaction), His6-GST (pDEST-565 in the LR reaction), or His6-MBP (pDEST-HisMBP in the LR reaction) tags appended to the Ntermini of the passenger proteins along with canonical TEV protease recognition sites (ENLYFQG) between the tags and the passengers (except for the vectors encoding TEV protease fusions, which contained the uncleavable recognition site ENLYFQP [21] instead). The pDEST-HisMBP derivative carrying an I329W mutation in.
