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.

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SummaryA generic protocol that utilizes a dual hexahistidine-maltose-binding protein (His 6 -MBP) affinity tag has been developed for the production of recombinant proteins in Escherichia coli. The MBP moiety improves the yield and enhances the solubility of the passenger protein while the His-tag facilitates its purification. The fusion protein (His 6 -MBP-passenger) is purified by immobilized metal affinity chromatography (IMAC) on nickel-nitrilotriacetic acid (Ni-NTA) resin and then cleaved in vitro with His 6 -tobacco etch virus protease to separate the His 6 -MBP from the passenger protein. In the final step, the unwanted byproducts of the digest are absorbed by a second round of IMAC, leaving nothing but the pure passenger protein in the flow-through fraction. Endogenous proteins that bind to the Ni-NTA resin during the first IMAC step also do so during the second round of IMAC. Hence, the application of two successive IMAC steps, rather than just one, is the key to obtaining crystallization-grade protein with a single affinity technique.

We describe a generic protocol for the overproduction and purification of recombinant proteins in Escherichia coli. The strategy utilizes a dual His 6 -maltose binding protein (HisMBP) affinity tag that can be removed from the target protein by digestion of the fusion protein at a designed site by tobacco etch virus protease. The MBP moiety serves to enhance the solubility and promote the proper folding of its fusion partners, and the polyhistidine tag facilitates its purification to homogeneity. This protocol is divided into three stages, each of which takes approximately 1 week to complete: (i) construction of a HisMBP fusion vector; (ii) a pilot experiment to assess the yield and solubility of the target protein; and (iii) the large-scale production and purification of the target protein.

Crystal structures of cleaved and uncleaved forms of the YscU cytoplasmic domain, an essential component of the type III secretion system (T3SS) in Yersinia pestis, have been solved by single-wavelength anomolous dispersion and refined with X-ray diffraction data extending up to atomic resolution (1.13 Å ). These crystallographic studies provide structural insights into the conformational changes induced upon auto-cleavage of the cytoplasmic domain of YscU. The structures indicate that the cleaved fragments remain bound to each other. The conserved NPTH sequence that contains the site of the N263-P264 peptide bond cleavage is found on a b-turn which, upon cleavage, undergoes a major reorientation of the loop away from the catalytic N263, resulting in altered electrostatic surface features at the site of cleavage. Additionally, a significant conformational change was observed in the N-terminal linker regions of the cleaved and noncleaved forms of YscU which may correspond to the molecular switch that influences substrate specificity. The YscU structures determined here also are in good agreement with the auto-cleavage mechanism described for the flagellar homolog FlhB and E. coli EscU.

Certain highly soluble proteins, such as Escherichia coli maltose-binding protein (MBP), have the ability to enhance the solubility of their fusion partners, making them attractive vehicles for the production of recombinant proteins, yet the mechanism of solubility enhancement remains poorly understood. Here, we report that the solubility-enhancing properties of MBP are dramatically affected by amino acid substitutions that alter the equilibrium between its "open" and "closed" conformations. Our findings indicate that the solubility-enhancing activity of MBP is mediated by its open conformation and point to a likely role for the ligand-binding cleft in the mechanism of solubility enhancement.Keywords maltose binding protein; solubility enhancer; affinity tag; fusion protein; solubility tag Proteins that normally accumulate in the form of insoluble aggregates when they are overproduced in E. coli can sometimes be recovered in a soluble, properly folded form if they are fused to a solubility-enhancing partner [1]. Consequently, the use of solubility-enhancing fusion partners has become an attractive alternative to the refolding of proteins. Many proteins have been reported to exhibit solubility-enhancing characteristics, including MBP, NusA, DsbA, thioredoxin, T7PK, Skp, SUMO, GB1 and the ZZ domain [2], although the evidence supporting these claims is stronger in some instances than in others. Yet the mechanism(s) by which certain soluble proteins enhance the solubility of their fusion partners remains poorly understood.E. coli MBP is an excellent solubility enhancer [3,4,5,6], but it is not the most effective affinity tag from the standpoint of protein purification [7,8]. Some fusion proteins do not bind efficiently to amylose resin, and even when they do, the purity of proteins after amylose affinity chromatography is usually inadequate. Two groups recently described mutations in MBP that increase its affinity for maltose [9,10]. Using a variety of experimental techniques, both groups reached the conclusion that these mutations exert their effect by altering the equilibrium between the "open" and "closed" conformations of MBP so as to favor the latter. In the open conformation, the ligand-binding cleft of MBP is exposed to solvent, whereas the closed conformation resembles that of the ligand-bound protein in which the cleft is largely buried.* Corresponding author, David S. Waugh, Ph.D., Tel: (301) 846-1842, Fax: (301) 846-7148, Email: waughd@ncifcrf.gov. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. We originally set out to determine whether these mutations wou...

SummaryInsolubility of recombinant proteins in Escherichia coli is a major impediment to their production for structural and functional studies. One way to circumvent this problem is to fuse an aggregation-prone protein to a highly soluble partner. E. coli maltose-binding protein (MBP) has emerged as one of the most effective solubilizing agents. In this chapter, we describe how to construct combinatorially-tagged His 6 MBP fusion proteins by recombinational cloning and how to evaluate their yield and solubility. We also describe a procedure to determine how efficiently a His 6 MBP fusion protein is cleaved by tobacco etch virus (TEV) protease in E. coli and a method to assess the solubility of the target protein after it has been separated from His 6 MBP.