The Ability to Enhance the Solubility of Its Fusion Partners Is an Intrinsic Property of Maltose-Binding Protein but Their Folding Is Either Spontaneous or Chaperone-Mediated

Abstract

Escherichia coli maltose binding protein (MBP) is commonly used to promote the solubility of its fusion partners. To investigate the mechanism of solubility enhancement by MBP, we compared the properties of MBP fusion proteins refolded in vitro with those of the corresponding fusion proteins purified under native conditions. We fused five aggregation-prone passenger proteins to 3 different N-terminal tags: His6-MBP, His6-GST and His6. After purifying the 15 fusion proteins under denaturing conditions and refolding them by rapid dilution, we recovered far more of the soluble MBP fusion proteins than their GST- or His-tagged counterparts. Hence, we can reproduce the solubilizing activity of MBP in a simple in vitro system, indicating that no additional factors are required to mediate this effect. We assayed both the soluble fusion proteins and their TEV protease digestion products (i.e., with the N-terminal tag removed) for biological activity. Little or no activity was detected for some fusion proteins whereas others were quite active. When the MBP fusions proteins were purified from E. coli under native conditions they were all substantially active. These results indicate that the ability of MBP to promote the solubility of its fusion partners in vitro sometimes, but not always, results in their proper folding. We show that the folding of some passenger proteins is mediated by endogenous chaperones in vivo. Hence, MBP serves as a passive participant in the folding process; passenger proteins either fold spontaneously or with the assistance of chaperones.

Dual Targeting of the Chemokine Receptors CXCR4 and ACKR3 with Novel Engineered Chemokines

“…For the first approach, we generated fusion constructs that in addition to CXCL12 inframe with the g3p coat protein included maltose-binding protein (MBP-CXCL12-g3p) or ubiquitin (Ubq-CXCL12-g3p) at the N terminus of CXCL12. Both maltose-binding protein and ubiquitin can function as solubilizing chaperones for otherwise insoluble proteins (42), including CXCL12 (43) (Fig. 1).…”

Development and Validation of a Robust and Sensitive Assay for the Discovery of Selective Inhibitors for Serine/Threonine Protein Phosphatases PP1α (PPP1C) and PP5 (PPP5C)

“…As a fusion partner, MBP improves the soluble yield of many heterologously expressed proteins in E. coli. 44 Indeed, our lab has had great success expressing ser/thr-specific protein phosphatases with good soluble yield in bacteria as MBP fusions. In our hands, the system works very well for expression of PP1a, PP2Ca, Wip1, and PP5C in an active form.…”

Bicistronic mRNAs to Enhance Membrane Protein Overexpression

“…The fusion protein can affect the folding state of the target protein, e.g., if too short linkers are used to connect both proteins [10]. Furthermore, the activity of the target protein can be compromised by the fusion [17][18][19], and the presence of a flexible fusion protein can interfere with crystallization [20].…”

“…Both types of fusions can be used to detect and even purify the target protein. Commonly used fusion proteins are MBP, the maltoside binding protein from E. coli [9,10], Trx (E. coli thioredoxin [11]), SUMO, (small ubiquitin-like modifier from S. cerevisiae, [12]), and Mistic (membrane integrating sequence for translation of inner membrane protein constructs from B. subtilis) [13]. In addition, novel fusion partners are constantly introduced, such as YnaI and YbeL, two small hydrophilic proteins from E. coli that have been recently reported to enhance the production of functional membrane proteins in E.…”