Nsively to circumvent inclusion body formation, particularly in E. coli where the poor solubility of recombinant proteins is a serious bottleneck [2,3,4]. However, the mechanism of fusionmediated solubility enhancement remains poorly understood. A variety of mechanisms, which are not necessarily mutually exclusive, have been proposed to explain how some but not all highly soluble proteins are able to function as solubility enhancers in the context of a fusion protein. One possibility is that solubility enhancers exert their effects by acting as “electrostatic shields”, reducing the probability of aggregation via electrostatic repulsion between highly charged soluble polypeptide extensions. While some solubility-enhancing fusion partners may function in this manner [5], this seems unlikely in the case of MBP because no correlation was observed between the net charges of MBPs from different microorganisms (all of which 1326631 share a very similar fold) and their 
efficacy as solubility enhancers [6]. Another possiblemechanism envisions the formation of soluble aggregates in which incompletely folded, hydrophobic passenger proteins occupy the center of a micelle-like sphere with hydrophilic domains shielding them from solvent. Indeed, there is good evidence for the formation of soluble, high molecular weight aggregates of human papilloma virus E6 fused to MBP [7]. How such seemingly “dead end” aggregates could evolve into properly folded fusion proteins remains unclear. Solubility enhancers have also been proposed to serve as “entropic anchors” by Fexinidazole chemical information restricting the motion of a slow folding passenger protein and enabling it to fold in a more entropically favorable environment by reducing the number of possible conformations that can be sampled [8]. If this theory is correct, then any soluble (and 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, 3687-18-1 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 Gateway cloning (Life Technologies Inc., Carls.Nsively to circumvent inclusion body formation, particularly in E. coli where the poor solubility of recombinant proteins is a serious bottleneck [2,3,4]. However, the mechanism of fusionmediated solubility enhancement remains poorly understood. A variety of mechanisms, which are not necessarily mutually exclusive, have been proposed to explain how some but not all highly soluble proteins are able to function as solubility enhancers in the context of a fusion protein. One possibility is that solubility enhancers exert their effects by acting as “electrostatic shields”, reducing the probability of aggregation via electrostatic repulsion between highly charged soluble polypeptide extensions. While some solubility-enhancing fusion partners may function in this manner [5], this seems unlikely in the case of MBP because no correlation was observed between the net charges of MBPs from different microorganisms (all of which 1326631 share a very similar fold) and their efficacy as solubility enhancers [6]. Another possiblemechanism envisions the formation of soluble aggregates in which incompletely folded, hydrophobic passenger proteins occupy the center of a micelle-like sphere with hydrophilic domains shielding them from solvent. Indeed, there is good evidence for the formation of soluble, high molecular weight aggregates of human papilloma virus E6 fused to MBP [7]. How such seemingly “dead end” aggregates could evolve into properly folded fusion proteins remains unclear. Solubility enhancers have also been proposed to serve as “entropic anchors” by restricting the motion of a slow folding passenger protein and enabling it to fold in a more entropically favorable environment by reducing the number of possible conformations that can be sampled [8]. 
If this theory is correct, then any soluble (and 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 Gateway cloning (Life Technologies Inc., Carls.
