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

Abstract

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...

Galectin-1 as a fusion partner for the production of soluble and folded human β-1,4-galactosyltransferase-T7 in E. coli

“…At times, for purification purposes, MBP-fusion protein does not bind to the amylose-affinity resin. Other times, after release from the affinity resin, protein precipitates out, possibly because the protein is misfolded or prone to aggregation in its native state [7, 10]. Here we show that another carbohydrate-binding protein, human galectin-1, can be an efficient fusion partner.…”

“…The best studied and most thoroughly validated solubility-enhancing protein is the maltose-binding protein (MBP). Structural studies of MBP have shown that it is an efficient enhancer of solubility only in the open conformation of the carbohydrate recognition domain (CRD) [10]. It is most likely that the CRD of the maltose binding protein is involved in folding of the passenger protein.…”

Positional effects of fusion partners on the yield and solubility of MBP fusion proteins

“…It has been proposed that MBP inhibits the formation of insoluble aggregates by transiently binding folding intermediates of an aggregation-prone passenger protein, effectively sequestering it in an intramolecular interaction that impedes the kinetically competing pathway of intermolecular aggregation and precipitation [4]. A corollary of this hypothesis is that MBP must be folded before it can bind to and sequester its passenger protein [6, 9]. If so, then as a result of co-translational folding, it follows that MBP should function as a more effective solubilizing agent when it is fused to the N-terminus of a passenger protein than to its C-terminus because in the former case MBP would emerge first from the ribosome and have time to fold before the passenger protein is translated.…”

Highly soluble and stable recombinant holo-phycocyanin alpha subunit expressed in Escherichia coli

“…Several mechanisms have been proposed to explain how MBP can enhance the solubility of the fusion protein [31,32]. It seems reasonable to assume that MBP can keep the proteins that it fused with in a properly folded form.…”