Gateway vectors for the production of combinatorially‐tagged His₆‐MBP fusion proteins in the cytoplasm and periplasm of Escherichia coli

Abstract

Many proteins that accumulate in the form of insoluble aggregates when they are overproduced in Escherichia coli can be rendered soluble by fusing them to E. coli maltose binding protein (MBP), and this will often enable them to fold in to their biologically active conformations. Yet, although it is an excellent solubility enhancer, MBP is not a particularly good affinity tag for protein purification. To compensate for this shortcoming, we have engineered and successfully tested Gateway destination vectors for the production of dual His 6 MBP-tagged fusion proteins in the cytoplasm and periplasm of E. coli. The MBP moiety improves the yield and solubility of its fusion partners while the hexahistidine tag (His-tag) serves to facilitate their purification. The availability of a vector that targets His 6 MBP fusion proteins to the periplasm expands the utility of this dual tagging approach to include proteins that contain disulfide bonds or are toxic in the bacterial cytoplasm.

Mutations that alter the equilibrium between open and closed conformations of Escherichia coli maltose-binding protein impede its ability to enhance the solubility of passenger proteins

“…It is possible that different solubility-enhancing proteins work by different mechanisms, or even by utilizing more than one mechanism, although comparative studies with MBP and E. coli NusA seem to suggest otherwise [12]. In any case, the results presented here are most consistent with the proposal that MBP posesses intrinsic chaperone-like qualities in the context of a fusion protein, and that the hydrophobic ligand-binding cleft plays a central role in its mechanism of action.…”

“…Next, the amplicons from these two PCRs were combined and used as the template for another PCR with primers PE-42 and PE-1425. The resulting PCR amplicon was digested with BglII and NotI, and then inserted between the unique BglII and NotI sites in pDEST-HisMBP [12] to generate an otherwise idential derivative of pDEST-HisMBP encoding the DM mutant MBP. Derivatives of pDEST-HisMBP encoding the I329W, W62E/Y155E, and W230E/W340E mutant MBPs were constructed with a QuickChange Site-Directed Mutagenesis kit (Stratagene).…”

“…The expression and solubility of various HisMBP fusion proteins was evaluated by standard procedures, using E. coli BL21(DE3) CodonPlus-RIL cells (Stratagene), as described [3,14,12]. All cultures were grown at 37 °C.…”

“…pDEST-HisMBP and its mutant derivatives produce the mature form of MBP with a hexahistidine tag joined to its N-terminus. The hexahistidine tag does not interfere with the ability of MBP to promote the solubility of its fusion partners [12]. However, its presence enabled us to purify all of the mutant MBPs examined in this study, some of which do not bind to amylose resin (see below), in precisely the same manner for biophysical characterization, using immobilized metal affinity chromatography.…”

Enhancing the Solubility of Recombinant Proteins in Escherichia coli by Using Hexahistidine-Tagged Maltose-Binding Protein as a Fusion Partner

“…Unfortunately, however, amylose affinity chromatography has proven to be problematic at times, not only because of the relatively low binding capacity of amylose resin for MBP but also because of persistent contaminants that require additional chromatographic steps to remove (5)(6)(7). We have demonstrated that a hexahistidine tag (His 6 ) can be added to the N-terminus of MBP without interfering with its ability to promote the solubility of its fusion partners (8). Therefore, IMAC can be used to circumvent the problems associated with amylose affinity chromatography.…”