Cloning and expression of recombinant human superoxide dismutase 1 (hSOD1) in Bacillus subtilis 1012

We aimed to clone and express the human Cu, Zn superoxide dismutase (hSOD1) in Bacillus subtilis 1012. Also, we investigated the expression level of hSOD1 under different induction conditions. As an essential member of the antioxidant defense system in vivo, hSOD1 has become a therapeutic agent against host diseases, such as oxygen toxicity, acute inflammation, and radiation injury. The recombinant hSOD1 was successfully secreted extracellularly into B. subtilis 1012. The expression conditions were optimized, including inoculum size, different media, temperatures, and inducer concentrations. Finally, the highest level of hSOD1 was produced as a soluble form in Super rich medium by 2% inoculum with 0.2 mM of IPTG at 37 °C after the induction for 24 h. Besides, 20 g/L of lactose also displayed the same inductive effect on hSOD1 expression as that of IPTG (0.2 mM). Finally, the specific activity of purified hSOD1 was determined to be 1625 U/mg in the presence of 800 μM of Cu2+ and 20 μM of Zn2+. We propose that the B. subtilis 1012-hSOD1 strain system has great potential in future industrial applications.


Introduction
Aerobic metabolism usually produces excessive reactive oxygen species (ROS), and organisms usually activate the antioxidant defense system to prevent ROS-induced damage in aerobic metabolism to maintain physiological homeostasis (Eleutherio et al. 2021;Neda Đorđević et al. 2018). 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 ). ROS will accumulate in abundance if the organism has a low SOD level, leading to DNA strand breakage, protein damage, or membrane lipid peroxidation (Kim et al. 2017).
All mammals contain 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 benefits 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 from living organisms is too low to meet the demand for clinical applications.
Researchers have developed efficient production systems for decades to achieve high expression of SOD, like engineering bacterial systems. In previous years, heterologous expression systems of human SOD (hSOD) were constructed, among which E. coli was the most commonly used one (Yang et al. 2020). 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 (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), P. 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 produced from E. coli expression system increased the production cost and thus limited its application to the large-scale expression of heterologous proteins . Compared to E. coli, yeast is a perfect expression system for post-translational protein modification and secretion. However, it has a relatively low secretion efficiency because it lacks a strong and strictly regulated promoter.
As a "Generally Recognized as Safe" (GRAS) organism, B. 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, B. subtilis can effectively overcome the shortcomings of E. coli and yeast expression systems, and about 60% of commercial enzymes are produced from B. subtilis (Huang et al. 2017). At present, hSODs were expressed in E. coli and P. pastoris. However, there is no report about their expression in B. subtilis.
In this study, we aim to construct a safe B. subtilis expression system of hSOD1 and explore the optimum expression condition for further industry study in the future. We cloned the encoding human sod1 gene into pHT43-His and transformed it into B. subtilis 1012. We investigated the expression level of recombinant hSOD1 with different media, temperatures, and inducers. Further, we examined the effect of Cu 2+ and Zn 2+ on the enzymatic activity of purified hSOD1.

Bacterial strains and plasmids
HepG2 cells (China Center for Type Culture Collection, Wuhan) were used for the hsod1 gene amplification. The initial cloning of hsod1 coding sequence was done through a T vector-pClone007 Blunt Simple Vector (Tsingke Biotechnology Co., Ltd., Beijing, China). E. coli DH5α competent cells (Tsingke Biotechnology Co., Ltd., Beijing, China) was used for subcloning and plasmid amplification. The expression vector used an E. coli-B. subtilis shuttle vector pHT43-His (Miaoling Biological Technology Co., Ltd., Wuhan, China), including a lacO operator and 6× His-Tag. The host strain B. subtilis 1012 (Lab collection) was applied for hSOD1 secretory expression. The cells were grown at 37 °C for 12 h in LB (10 g/L of Tryptone, 5 g/L of Yeast extraction, 10 g/L of NaCl, pH 7.0), Super Rich (25 g/L of Tryptone, 20 g/L of Yeast extraction, 3 g/L of K 2 HPO 4 , and 30 g/L of Glucose, pH 7.0), or 2× YT (16 g/L tryptone, 10 g/L yeast extract, 5 g/L NaCl, pH 7.0) medium. Antibiotics for clone selections were as follows: 100 µg/mL Ampicillin for E. coli; 5 µg/mL Chloramphenicol (Cm) for B. subtilis.

Construction of expression plasmid
The hsod1 gene with the partial vector sequence was amplified using KOD-Plus-Neo (Toyobo, Osaka, Japan) from pClone007 Blunt Simple Vector-hSOD1. The recombinant primers were composed of SOD1-pHT43-His-F: ACA TCA GCC GTA GGA TCC ATG GGA TCC ATG GCG ACG AAG GCC GTGT and SOD1-pHT43-His-R: TAG CGG CCG CAT GGA TCC TTG GGC GAT CCC AAT TAC AC, in which the underlined sequences were the partial pHT43-His vector sequence. The pHT43-His plasmid was digested and linearized with BamHI (TaKaRa Biotech Co., Ltd, Shiga, Japan) and AP (Alkaline Phosphatase) by MonClone TM Fast AP (Monad Biotech Co., Ltd, Wuhan, China). The purified gene product was inserted into a restricted pHT43-His for homologous recombination using the MonClone TM Hi-Fusion Cloning Mix V2 (Monad Biotech Co., Ltd, Wuhan, China). The constructed plasmid was transformed into E. coli DH5α competent cells, and the positive transformants were screened by colony PCR and sequenced.
Preparation and transformation of competent B. subtilis 1012 The confirmed recombinant plasmid pHT43-His-hSOD1 was transformed into B. subtilis 1012 according to the Spizizen method (Spizizen 1958). B. subtilis 1012 was inoculated on LB plate and incubated at 37 °C for 12 h. Then, the host strain was transferred into 2 mL LB medium using a loop and cultured under 180 rpm of shaking at 28 °C overnight. Next, 50 µL bacterial culture was inoculated in 5 mL SP I medium (195.2 mL SP, 0.1 g casein, 0.2 g yeast, 1 g glucose, 4.8 mL ddH 2 O) under 180 rpm of shaking at 37 °C. The growth curve was measured every 30 min until the plateau phase. After that, 0.5 mL of the culture was added to 4.5 mL of SP II medium (99 mL SP I, 0.05 g MgCl 2 , 0.007 gCaCl 2 , 1 mL ddH 2 O) and incubated under 100 rpm of shaking at 37 °C for 90 min. And 50 µL EGTA (100 mM) was added to the culture medium and incubated under 100 rpm of shaking at 37 °C for 10 min. Finally, the culture was dispensed with 300 µL for each tube (Brockmeier et al. 2006;Murayama et al. 2004).
For the bacterial transformation, 1 µg pHT43-His and pHT43-His-hSOD1 were mixed with B. subtilis 1012 competent cells and cultured under 100 rpm of shaking at 30 °C for 90 min. Then, the bacterial culture was inoculated on LB plates containing 5 µg/mL Cm at 37 °C.
Western blotting of hSOD1 in B. subtilis 1012 with different inducer concentrations, induction times, and temperatures The hSOD1 was induced with different IPTG concentrations (0.2−1mM) at 37 °C for 12−60 h. Also, the recombinant protein was collected at different temperatures after induction of 0.2 mM IPTG for 24 h. And finally, the hSOD1 expression was assessed by the induction with 20 g/L lactose at 37 °C for 12−72 h. Next, a 20 µL denatured sample was separated on a 15% SDS-PAGE for Western blot analysis. After the electrophoresis, the gels were visualized by Coomassie brilliant blue R-250 staining and then transferred to polyvinylidene difluoride (PVDF) membranes. A mouse-anti His-Tag mAb (1:2000, Cat #AE003) (ABclonal, Wuhan, China) was used to detect the FLAG-tagged recombinant hSOD1. The goat anti-mouse second antibody (1:5000) (Cell Signaling Technology, MA, USA) was used to detect the native form of hSOD1 with His-tag. Enhanced chemiluminescence (ECL) (Sigma Aldrich, MO, USA) was used to detect the protein bands. The expression levels of hSOD1 under different induction conditions were analyzed using Image J software (NIH, Bethesda, MD, USA). The 15% SDS-PAGE was run at 100 V for 3 h at 4 °C to avoid protein denaturation.
Purification and enzyme activity assay of hSOD1 in B. subtilis 1012 The B. subtilis 1012 containing pHT43-His-hSOD1 was pre-cultured in LB at 37 °C overnight (approximately 12 h) and then incubated in Super rich medium (5 µg/mL Cm) with 2% inoculum size. The recombinant hSOD1 was induced by 0.2 mM IPTG at 37 °C for 24 h. Then, the cellular supernatant was collected and loaded onto a 5-mL HisTrap TM FF nickel Sepharose column (GE Healthcare) for purification using an AKTA pure chromatography system (GE Healthcare). After the His-tagged target protein was separately eluted with 20 and 500 mM of imidazole, the hSOD1 fractions were pooled and desalted with a 5-mL HiTrap TM Desalting column (GE Healthcare). Protein concentration was measured with a BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA). The purified protein was detected as described above.
The activity of purified hSOD1 was detected in vitro using a Total Superoxide Dismutase Assay Kit with WST-8 (Beyotime Institute of Biotechnology, Shanghai, China). In brief, 20 μL hSOD1, 160 μL WST-8/enzyme working solution, and 20 μL reaction starting solution were mixed and incubated at 37 °C for 30 min. After the reaction was stopped, the absorbance was detected at 450 nm based on the color change of a water-soluble formazan dye. One unit of SOD was defined as the enzyme amount in a 20 μL sample that inhibited the reduction reaction between WST-8 and superoxide anion by 50%. The purified hSOD1 activity was normalized in the presence of Cu 2+ and Zn 2+ with different concentrations against that without Cu 2+ and Zn 2+ .

Statistical analysis
Data were presented as mean ± SD. Statistical analysis was carried out with IBM SPSS Statistics 25. The statistical differences were determined using one-way analysis of significance (ANOVA). Values of P < 0.05 were considered statistically significant.

Growth of recombinant 1012-hSOD1 strain in different culture media
To explore the effects of media and bacterial inoculum on the growth of recombinant 1012-hSOD1 strain, we cultured the bacteria in three different media containing 1% or 2% inoculum at 37 °C. The maximum growth was observed by OD 600 values at other time points. As shown in Fig. 1, either 1% or 2% inoculum led to faster growth of 1012-hSOD1 strain in Super rich media than LB or 2× YT media ( Fig. 1a and 1b). There was no difference in the bacterial growth between LB and 2× YT media with 1% and 2% inoculum (Supplementary Fig. 1a and 1b). The strain grew optimally at 2% inoculum for the Super rich medium compared to 1% inoculum (Supplementary Fig. 1c). Thus, the Super rich media with 2% inoculum was chosen for the recombinant hSOD1 protein expression.
Secretory expression of hSOD1 in B. subtilis 1012 using different IPTG concentrations, induction times, and temperatures The recombinant protein expression is usually affected by IPTG concentration, induction time, and temperature. IPTG is an efficient inducer for the expression system with lac promoter, which can't be metabolized by bacteria and is hence stable. We next investigated the effect of IPTG concentration on hSOD1 expression at 37 °C. The 1012-hSOD1 strain samples in the supernatant of Super rich medium were separately collected at 6, 12, 24, 36, 48, and 60 h after a series of IPTG concentrations (0.2, 0.4, 0.6, 0.8, and 1 mM) were chosen, respectively. And the highest expression efficiency of hSOD1 was obtained after the induction with different concentrations of IPTG for 24 h. After that, the hSOD1 expression decreased gradually (Fig. 2a-2e). The optimal IPTG concentration was determined to be 0.2 mM, which led to a higher expression level of hSOD1 than other IPTG concentration groups (P < 0.05) (Fig. 2f).
Since the induction temperature is also vital to enhance recombinant protein expression, the strain 1012-hSOD1 was induced by 0.2 mM IPTG for 24 h at 25, 30, 37, and 40 °C, respectively. As suggested in Fig. 3, the expression level of hSOD1 at 37 °C was significantly higher than other temperature groups (P < 0.05).
Secretory expression of hSOD1 in B. subtilis 1012 induced by lactose As indicated in Supplementary Fig. 2, we explored the effect of lactose concentration on hSOD1 Fig. 1 Growth curve of recombinant pHT43-His-hSOD1 strain in different media and inoculum. a OD 600 of 1012-hSOD1 strain cultured in Super rich, LB, or 2× YT media with 1% inoculum. b OD 600 of 1012-hSOD1 strain in Super rich, LB, and 2× YT media with 2% inoculum expression at 37 °C. It was suggested that the optimal concentration of lactose was 20 g/L. Further, our studies revealed that the expression of hSOD1 reached the maximal level after the induction for 24 h with 20 g/L of lactose (Fig. 4a). Noticeably, 20 g/L of lactose displayed the same inductive effect on hSOD1 expression as that of 0.2 mM IPTG (Fig. 4b).
Purification and enzyme activity of recombinant hSOD1 Next, we purified hSOD1 using a Nickel column, performed by chromatography (chromatographic) system on the AKTA pure 25 (GE Healthcare Life Sciences, Uppsala, Sweden). Under the optimized induction conditions, the hSOD protein was collected from 1 L fermentation broth. After the purification, elution, and desalination, the recombinant protein was detected by SDS-PAGE (Fig. 5).
The effects of Cu 2+ and Zn 2+ metal ions on hSOD activity were investigated. As shown in Fig. 6, a 51.8fold increase in the specific activity of hSOD1 was observed after the supplementation of Cu 2+ (200 μM) alone, whereas 5 μM of Zn 2+ alone slightly increased the SOD activity. Interestingly, the hSOD1 activity elevated about 160-fold after the supplementation with Cu 2+ (200 μM) plus Zn 2+ (5 μM) compared to the control. And the highest activity was obtained after the combined use of 800 μM of Cu 2+ plus 20 μM of Zn 2+ , which induced a 1300-fold increase in enzymatic activity, which displayed a significant difference compared to other experimental groups (P < 0.05) (Fig. 6).

Discussion
The occurrence of many diseases is related to the deficiency or false folding of SOD1, such as amyotrophic lateral sclerosis, inflammatory bowel diseases, and lung cancer (Dziabowska-Grabias et al. 2021;Proctor et al. 2016;Wang et al. 2021). In addition to diminishing oxidative stress and ROS generation, SOD1 can inhibit the activation of endothelial cells and regulate the expression of adhesion molecules (Carroll et al. 2007;Segui et al. 2004). Recently, SOD1 was also reported to facilitate cytoprotective pathways by activating gene transcription and play a physiological role in regulating signal transductions involving ROS, indicating a high potential in disease treatment (Trist et al. 2021). These biological activities make SOD1 become a potential therapeutic agent against host diseases.
A variety of literatures have reported the heterologous expression of hSOD1 in different expression systems. A high level and activity of hSOD1 was found in E. coli, by which 1 L fermentation broth produced 342 mg of purified hSOD1 with an enzymatic activity of 46,541 U/mg (Yang et al. 2020). In transgenic tobacco, 40 ng/mg of hSOD1 was obtained from fresh leaf tissues (Park et al. 2002). And the activity of hSOD reached 1451 ± 136 U/mL in goat milk (Lu et al. 2018). Though the hSOD1 expression level was not the highest (36 mg/L and 1625 U/mg) in this study compared with previous reports, the B. subtilis system was more suitable for industrial production from the perspective of safety and operation simplicity. B. subtilis has been widely applied to agriculture, medicine, and industry. In addition to induction conditions, the modification of B. subtilis can also improve the production of recombinant proteins using promoter elements (Le et al. 2022). In future work, these factors need to be considered.
IPTG has been commonly used to induce the expression of recombinant proteins in E. coli and B. subtilis. Previously, 1 mM IPTG was usually used to induce protein expression (Tran et al. 2017). Our studies demonstrated that a low concentration of IPTG (0.2 mM IPTG) had a better inductive effect (Fig 2f). This can be explained by the fact that high concentrations of IPTG are harmful to host cells (Larentis et al. 2014). In this study, the host B. subtilis 1012-hSOD1 strain shuttles a vector pHT43-His, which had a strong IPTG-inducible Pgrac promoter Mean values with the same letters (also including a and ab, ab and bc, bc and c) are not significantly different (P ≥ 0.05) ◂ with a lac operator. Thus, the recombinant hSOD1 can be expressed by IPTG or lactose as inducers. . Compared to IPTG, lactose is a safer and cheaper inducer. In this study, lactose (20 g/L) had similar inductive activity to that of IPTG (0.2 mM) in the expression of hSOD1 (Fig. 4b), and the heterogeneous hSOD1 can be directly obtained from the supernatant of the culture medium. Based on the above, our detection system not only saves the purification cost but also avoids possible activity loss due to protein purification. These advantages lay a foundation for the future industrial production of hSOD1.
The sod1 gene is located on chromosome 21q22.11 and encodes Cu/Zn superoxide dismutase (Abati et al. 2020). Human SOD1 protein forms a homodimer, and each monomer binds Cu 2+ and Zn 2+ to harbor a disulfide. Both metal cofactors are necessary for catalyzing one-electron oxidation followed by oneelectron reduction of two O 2 •anions to affect disproportionation (Griess et al. 2017). SOD1 exerts its catalytic function when it binds a Cu 2+ and a Zn 2+ per molecule to form an intramolecular disulfide bridge (Banci et al. 2011). Without the involvement of Cu 2+ , Zn 2+ alone failed to guarantee the correct folding of hSOD1, leading to its activity loss or even degradation (Rumfeldt et al. 2009). In this study, we purified hSOD1 and detected the highest activity when 800 μM of Cu 2+ and 20 μM of Zn 2+ were used for catalytic reaction (Figs. 5 and 6). By comparing the optimal catalytic concentrations between Cu 2+ and Zn 2+ , we observed that Cu 2+ played a predominant role in hSOD1 activity, and a minor contribution was Mean values with the same letters are not significantly different (P ≥ 0.05) made by Zn 2+ in the hSOD1-mediated catalysis, in consistency with a previous report (Li et al. 2010). Our results proved that the synergistic effect of Cu 2+ and Zn 2+ was essential for the exertion of hSOD1 activity.
Since E. coli is a conditional pathogen, the products from E. coli expression systems must be extracted and purified before applying to human health. Different from E. coli, B. subtilis is a safe probiotic in mammals. Both the bacterium itself and its expression products can be directly used for disease treatment. It was suggested that SOD1 produced from genetically engineered lactic acid bacteria could be applied to treat ROS-induced gastrointestinal pathologies (Zhang et al. 2013). In another study, a catalaseor SOD-producing lactic acid bacteria increased the degradation of H 2 O 2 and reduced the severity of colitis (LeBlanc et al. 2011). In our studies, the highest expression of hSOD1 was achieved after the induction for 24 h at 37 °C (Fig 3), which is close to body temperature. This makes B. subtilis 1012-hSOD1 strain a possibility for drug therapy in vivo.

Conclusion
In summary, we established a B. subtilis expression system with hSOD1 activity. The hSOD1 was induced by 0.2 mM of IPTG at 37 °C for 24 h in Super rich medium. By nickel affinity chromatography, we obtained the purified hSOD1 with a concentration of 36 mg/L. The specific activity of purified hSOD1 was determined to be 1625 U/mg in the presence of 800 μM of Cu 2+ and 20 μM of Zn 2+ . Besides, the hSOD1 expression in B. subtilis can be easily induced by lactose. Our studies shed light on the future industrial production of engineering bacteria 1012-hSOD1.
Author contributions Hongtao Liu and Zhigang Zhang designed the study. Mingzhu Yin, Nian Wang, Qiqi Wang, Hui Xia, Xue Cheng, and Haiming Hu were responsible for the acquisition of data. Mingzhu Yin and Nian Wang interpreted the experimental data. Mingzhu Yin and Hongtao Liu were the major contributors in drafting and revising the manuscript. All authors have read and approved the final manuscript.