Aerobic metabolism usually produces excessive reactive oxygen species (ROS). To maintain the physiological homeostasis, organisms will activate the antioxidant defense system to protect cells against ROS-induced damage in aerobic metabolism. In a previous report, superoxide dismutase (SOD) was proved to be the first line of in vivo defense against ROS(Yang et al., 2020). SOD catalyzes the dismutation of superoxide radicals to produce oxygen and hydrogen peroxide(Nguyen et al., 2020). If the organism has a low SOD level, ROS will accumulate in abundance, leading to DNA strand breakage, protein damage, or even membrane lipid peroxidation (Kim et al., 2017).
All mammals possess three members of SOD with tightly regulated localization patterns. Of these, there are two copper/zinc superoxide dismutases (Cu, Zn-SODs), i.e., SOD1 and SOD3. SOD1 exists in the cytosol, mitochondrial inter-membrane space, and nucleus, while SOD3 is an extracellular dismutase. Both SODs play a pivotal role in the antioxidant defense system. The manganese-containing superoxide dismutase (MnSOD, SOD2) localizes in the mitochondrial matrix. It is beneficial in the occurrence of many diseases like atherosclerosis (Eleutherio et al., 2021). Thus, SOD is a crucial contributor to alleviating the harmful effects of ROS. However, the physiological level of SOD is too low to meet the demand of clinical applications from living organisms.
For decades, researchers have developed new strategies have to achieve high-yield SOD by using efficient production systems like engineering bacterial systems. In previous years, heterologous expression systems of human SOD (hSOD) were constructed, and E coli was the most commonly used one. To date, the recombinant hSOD1 has been expressed in the cytosol of E. coli strain A1645 and E.coli BL21 (DE3) (Hartman et al., 1986; Lin et al., 2018; Yang et al., 2020). Also, hSOD1 was produced in eukaryotic cells, such as Pichia pastoris (Park et al., 2002; Wu et al., 2009), insect cells (Hayward et al., 2002), plant cells (Park et al. 2002), and mammary glands of transgenic animals (Lu et al., 2018). Besides, the full-length hSOD2 recombinant protein was obtained in E.coli Rosetta-gami, BL21 (DE3), and mammalian cells (Hosoki et al., 2012; Pan et al., 2017). Also, hSOD3 was expressed in E.coli (Bae et al., 2013), Pichia pastoris (Chen et al., 2006), insect cells (He et al., 2002), and Chinese hamster ovary cells (Tibell et al., 1987). However, the above systems have their limitations in protein production. For example, the inclusion bodies and endotoxins formed in E.coli expression system increased the production cost and thus limited its application to the large-scale expression of heterologous proteins (K. Zhang et al., 2017). Although the yeast is a perfect expression system for the post-translational protein modification and secretion compared to E.coli, it has a relatively low secretion efficiency for the lack of a strong and strictly regulated promoter.
As a “generally Recognized as Safe” (GRAS) organism, Bacillus subtilis is nonpathogenic and has developed into an attractive host, with no apparent bias of codon usage, especially in the secretion of extracellular functional proteins into the culture medium. This can simplify the purification process of protein and provide a correctly folded and soluble heterologous protein (Gu et al., 2018; Huang et al., 2017; Niu et al., 2018). Collectively, Bacillus subtilis can effectively overcome the shortcomings of E.coli and yeast expression systems. Nowadays, about 60% of commercial enzymes are produced from Bacillus subtilis (Huang et al., 2017). Although hSODs have been expressed in Escherichia coli and Pichia pastoris, there is no report about their expression in Bacillus subtilis.
This study cloned the encoding human sod1 gene into pHT43-His and transformed it into Bacillus subtilis 1012. We investigated the expression level of recombinant hSOD1 with different media, temperatures, and inducers. Further, we examined the effect of Cu2+ and Zn2+ on the enzymatic activity of purified hSOD1.