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.

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

A model illustrating the roles that MBP plays in the production of recombinant proteins (see text for discussion).

<p>A model illustrating the roles that MBP plays in the production of recombinant proteins (see text for discussion).</p

Primer sequences.

<p>Primer sequences.</p

Interaction of MBP fusion proteins with GroEL/S.

<p>(<b>A</b>) Lysed cells co-expressing H₆-MBP-GFP and either wild-type GroE or the GroE_3–1 variant are shown under blue or white light illumination. Cells co-expressing GroE_3–1 fluoresce more intensely than cells co-expressing wild-type GroE as a result of enhanced GFP folding. Cells expressing only the MBP-GFP fusion protein are shown on the left. (<b>B</b>) SDS-PAGE analysis of total and soluble proteins from the cells in (A). T, total intracellular protein; S, soluble intracellular protein.</p

Design of fusion proteins.

<p>Schematic representation of fusion proteins with three different N-terminal tags: H₆, H₆-GST, and H₆-MBP (not to scale). In the tagged forms of TEV protease, the canonical TEV protease recognition site (ENLYFQG) was replaced by an uncleavable recognition site ENLYFQP <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049589#pone.0049589-Kapust3" target="_blank">[21]</a> to prevent autodigestion of the fusion proteins.</p

The addition of GroEL and GroES increases the yield of properly folded passenger proteins <i>in vitro</i>.

<p>(<b>A</b>) G3PDH activity. (<b>B</b>) DHFR activity.</p

Specific activity of refolded vs. natively purified fusion proteins.

<p>Specific activity of refolded vs. natively purified fusion proteins.</p