A variety of fusion tags are used to increase expression yields and change solubility and native folding . However, it is still not clear how fusion tags enhance protein expression. Ub was reported to exert chaperoning effects on fusion proteins, thus increase expression of proteins in E. coli and yeast [7, 16]. Indeed, Ub has a highly stable structure and is the fastest folding protein known . Thus, it may serve to stabilize and promote proper folding of the fusion target. SUMO, structurally similar to Ub, also promotes folding and structural stability of fusion proteins, and leads to their enhanced expression [18–20].
Prokaryotic MoaD and ThiS share the structure of Ub-like domain with their eukaryotic counterparts Ub and SUMO. Our current results indicate that MoaD confers the enhanced expression of EGFP, either in N-terminal fusion or in C-terminal fusion. In contrast to eukaryotic counterparts Ub and SUMO, MoaD fusion at both N- and C-terminus reduces the soluble protein expression rather than enhances the solubility. This is the same case for ThiS fusion. Instead of promoting its proper folding in refolding experiments, MoaD hinders the native EGFP folding, in a similar way to ThiS. This was not expected because they are stable small proteins with a similar conserved structure as Ub or SUMO. The slowdown of folding, rather than the fast expression of fusion protein, should be a primary factor to drive the expressed fusion protein to inclusion bodies. This was proved by the fact that the most of EGFP with MoaD fusion at C-terminus are aggregated in inclusion bodies as detected by confocal microscopy. Actually, the fast expression of fusion protein should not occur under the leaky expression condition.
Enhancing the production of inclusion bodies is one of the fusion strategies although fewer tags are reported for this purpose . Inclusion bodies protect the products from proteolyses, and usually lead to a higher expression yield. In this study, MoaD did not drive the satisfied overexpression of insulin A and B chains in comparison to the ThiS fusions because of a decreased ability of MoaD in driving the protein aggregation of insulin chains.
Different fusion systems have given variable results of expression [1, 3, 4], and no single fusion tag is ideal for every protein target. On the expression of unstable mRI, MoaD fusion affords complete protection of the product from intracellular degradation. Ub- and SUMO-fusion of mRI, although leading expression products to inclusion bodies, show a moderate intracellular degradation of products . This difference may be attributed to the rapid aggregation of MoaD fusion product to inclusion bodies that afford protection from proteolytic degradation. On the other hand, ThiS fusion, expected to enhance the rapid aggregation due to the sluggish of folding in a similar way as MoaD fusion, led to the significant degradation of mRI. An active ThiS-directed degradation was anticipated for this unusual observation .