Currently, the foreign protein expression systems include the prokaryotic E. coli system used in this study and the eukaryotic systems such as yeast, Baculovirus, and mammalian cells (Phan et al. 2014; Smith et al. 1983; Berting et al. 2010). The high cost and long culture cycle of eukaryotic expression systems greatly restrict their application, so prokaryotic E. coli is still the most promising strain for producing recombinant proteins. It can’t modify the protein expression after translation, enabling the formation of the inclusion body protein (Stewart et al. 2014). Several studies have demonstrated that the SM nuclease in E. coli is expressed in the form of inclusion bodies (Zhao. 2017; Cai. 2011). Another study has reported that the soluble recombinant SM nuclease can be expressed in E. coli, with low expression and yield (Ma et al. 2022).
This study revealed that the Sumo-fused SM nuclease possessed a significantly higher expression compared with that without SUMO-fusion. Furthermore, the expression of inclusion body obtained in this study was higher in contrast to that acquired previously (Zhao 2017; Cai 2011). SUMO tag in E. coli enables high expression of the target protein by correctly folding it (Butt et al. 2005). In addition, SUMO fusion not only elevates the expression of the target protein, but also enhances the soluble form of its expression (Munir et al. 2023; Takahashi et al. 2023). However, no soluble expression of SM nuclease after SUMO fusion was observed in this study under the same expression conditions. To investigate the role of SUMO fusion in elevating the soluble expression of SM nuclease, relevant experiments were carried out by varying the induction temperature and IPTG concentration. Three induction temperatures (16℃, 25℃, and 42℃) were selected and the final concentration of IPTG was determined as 0.5 mM. Meanwhile, the induction duration was designated as 3 h. The expression experiments were performed at two different IPTG concentrations (0.1 mM and 1 mM) and an induction duration of 3 h at 37℃. The results disclosed that there was no expression protein band at 29 kDa in the supernatant solution of SM nuclease under all conditions (Fig. 8). Furthermore, there existed no expression protein band at 45 kDa for the supernatant digested from the SUMO-fused SM nuclease under any conditions. Consequently, the two SM nucleases constructed on the pET28a vector could not achieve soluble expression, providing no support for assessing the effect of SUMO fusion in elevating the soluble expression of the SM nuclease.
Since SM nuclease is prone to be expressed in an insoluble form, acquiring an active protein through denaturation and renaturation becomes essential. This study indicated that the SUMO tag increased the solubility of the inclusion body protein under the same washing conditions, enhancing the capability of impurity removal, which was better than that obtained in previous research (Zhao 2017; Cai 2011). This phenomenon may be partly attributed to the special structure of the SUMO protein, which contains an outer hydrophilic surface and an inner hydrophobic core (Butt et al. 2005; Malakhov et al. 2004).
Protein refolding is not only a challenging and time-consuming task, but also is hindered by a lack of clear understanding regarding the underlying mechanism, making the success of the refolding uncertain. During the refolding, an intermediate product is formed at an early stage, which is prone to aggregation. Correct folding of the disulfide bond is key to successful refolding, and the formation of misfolded disulfide bonds leads to protein precipitation. As a result, different outcomes were observed in this experiment. Without SUMO fusion, some SM nuclease proteins were misfolded and precipitated. In contrast, a SUMO fusion, the refolding success rate was significantly improved. This is because the folded SUMO can serve as a universal molecular chaperone to prevent aggregation of the intermediate, allowing them to remain in solution long enough to adopt the correct conformation (Mayer et al. 2004; Burgess et al. 2009).
In this study, the HPLC results demonstrated that the purity of the SUMO-fused SM nuclease obtained was up to 99%. In contrast, the purity of the SM nuclease obtained was reported as only 85% (Cai 2011) or 85% (Zhao 2017). In this study, purification of inclusion body was combined with the SUMO fusion expression. During the inclusion body wash, some of the impurity proteins can be removed firstly, followed by further purification by two affinity chromatography steps. In 2020, Zhou et al. introduced a new purification method of SUMO and achieved a purity of the protein of only 80%. The comparison suggests that the purification method adopted this study represents a significant improvement, even though it involves multiple purification steps.
Finally, this study unveiled that the activity of the SUMO-fused SM nuclease exhibited significantly higher activity compared to the unfused SM nuclease. It could reach 4 × 106 U/mg, suppressing the results obtained in the previous studies (Zhao 2017; Cai. 2011; Ma et al. 2022). Its activity is obviously associated with two pairs of disulfide bonds, namely C9 and C13 at the N-terminus and C201 and C243 at the C-terminus of SM nuclease. Therefore, Alphafold2 was selected to predict the structure of the SM nuclease after SUMO fusion in this study (Fig. 9a). As illustrated in Fig. 9b, the two SM nucleases had similar tertiary structures. However, the interaction force between the two pairs of disulfide bonds of the SUMO-fused SM nuclease was more abundant in comparison to that of the unfused SM nuclease. This enhanced interaction force may be the mechanism by which the SUMO tag assists in the successful refolding of the target protein. The fusion expression altered the interaction force of the amino acids at the disulfide bond (Fig. 10), thereby increasing the probability of protein refolding.
In summary, this study demonstrated that SM nucleases successfully increased their expressions through SUMO fusion, enhanced protein solubility during inclusion body washing, and improved protein renaturation efficiency during renaturation. However, the role of SUMO fusion in elevating the soluble expression of SM nuclease was not observed. After the inclusion body was washed and purified once, the purity of protein could reach 99% by double affinity chromatography, representing a substantial improvement. Activity of the final SM nuclease reached 4×106 U/mg. The mechanism governing the correct folding of the protein during renaturation of the SUMO tag remains a subject to be investigated. In conclusion, SUMO fusion has proven to be an effective heterologous protein expression system. SUMO offers advantage in facilitating easy separation and purification of inclusion bodies as well as promoting effective chaperone protein refolding in vitro. By calculation, this work yielded approximately 10 mg of SM nuclease protein with a purity of 99% from 1 g of bacteria using SUMO fusion, which greatly accelerated the production of recombinant SM nuclease. Meanwhile, this method provides an important basis for protein expression and purification of other forms of inclusion bodies.