Purification and characterization of recombinant human superoxide dismutase integrated with resilin-like polypeptide.

Human superoxide dismutase (hSOD1) plays an important role in the aerobic metabolism and free radical eliminating process in the body. However, the production of existing SOD faces problems such as complex purification methods, high costs, and poor product stability. This experiment achieved low-cost, rapid, and simple purification of hSOD1 through ammonium sulfate precipitation method and heat resistance of recombinant protein. We constructed a recombinant protein hSOD1-LR containing a resilin-like polypeptide tag and expressed it. The interest protein was purified by ammonium sulfate precipitation method, and the results showed that the purification effect of 1.5 M (NH4)2SO4 was the best, with an enzyme activity recovery rate of 80 % after purification. Then, based on its thermal stability, further purification of the interest protein at 60 °C revealed a purification fold of up to 24 folds, and the purification effect was similar to that of hSOD1-6xHis purified by nickel column affinity chromatography. The stability of hSOD1-LR showed that the recombinant protein hSOD1-LR has better stability than hSOD-6xHis. hSOD1-LR can maintain 76.57 % activity even after 150 min of reaction at 70 °C. At same time, hSOD1-LR had activity close to 80 % at pH < 5, indicating good acid resistance. In addition, after 28 days of storage at 4 °C and 40 °C, hSOD1-LR retained 92 % and 87 % activity, respectively. Therefore, the method of purifying hSOD1-LR through salt precipitation may have positive implications for the study of SOD purification.

Characterization of a novel extracellular CuZn superoxide dismutase from Rimicaris exoculata living around deep-sea hydrothermal vent.

Superoxide dismutase (SOD, EC 1.15.1.1) is a member of metalloenzyme that plays a key role in protecting organisms from oxidative damage. A novel extracellular CuZn superoxide dismutase RESOD was identified from Rimicaris exoculata, a dominant species that lives in close proximity to the deep-sea hydrothermal vents. It encoded a protein consisting of 227 amino acids with a signal peptide of 22 amino acids. Sequence analysis revealed that it had the characteristics of CuZn superoxide dismutase, and had low homology with the known SODs. Then the recombinant RESOD was expressed successfully, and high-purity RESOD was obtained. The recombinant RESOD exhibited maximal activity and stability with a temperature range of 0 °C to 10 °C. And the optimal pH for the activity and stability was about 10. However, RESOD was sensitive to some metal ions, particularly calcium. Furthermore, the biological function of RESOD was investigated in HeLa cells. It was found that RESOD could reduce the level of oxidation, and decrease the apoptosis resulted from excessive oxidant challenge. In conclusion, a novel alkali-tolerant cold-active extracellular CuZn SOD was characterized. The characteristics make RESOD a good candidate in a wide range of applications.

A novel, kinetically stable copper, zinc superoxide dismutase from Psychropotes longicauda.

Superoxide dismutases (SODs) are one of the most important antioxidant enzymes against oxidative damage. In the present study, we cloned and expressed a novel and stable Cu, Zn-SOD from a hadal sea cucumber Psychropotes longicauda (i.e., Pl-Cu, Zn-SOD). The purified recombinant enzyme was intracellular, dimeric with the Mr. of approximately 38 kDa, with the expressed activity from 0 °C to 60 °C at an optimal temperature of 20 °C and 30 °C and maximum activity at the pH of 8.0. The Km and Vmax values of Pl-Cu, Zn-SOD were 0.041 ±â€¯0.004 mM and 1450.275 ±â€¯36.621 U/mg, respectively. At tested conditions, Pl-Cu, Zn-SOD was relatively stable in chemicals, such as ß-ME, EDTA, Tween 20, Triton X-100, and Chaps, especially in urea and guanidine hydrochloride, which can resist protease hydrolysis and tolerate high hydrostatic pressure of 100 MPa and 2 M NaCl. All these properties make Pl-Cu, Zn-SOD a candidate in the biopharmaceutical and nutraceutical fields, and help us better understand the adaptation mechanism of hadal area.

High-Throughput Microplate-Based Fluorescence Assays for Studying Stochastic Aggregation of Superoxide Dismutase-1.

Investigating in vitro kinetics of superoxide dismutase-1 (SOD1) aggregation with high-throughput microplate-based assays provides valuable information regarding SOD1 pathogenesis in amyotrophic lateral sclerosis (ALS) and opens venues for the development of effective therapies. In this chapter, we first explain the step-by-step purification and demetallation of wild-type (WT) and ALS-variant SOD1 proteins from Saccharomyces cerevisiae (baker's yeast). We then describe the methodology for a microplate-based fluorescence assay that is used to study real-time kinetics of metal-free (apo)-SOD1 aggregation. This technique is highly sensitive, semiautomated, requires minimum modifications to protein, and produces a plethora of data in a short period of time. We also describe a new approach for extracting clinically relevant information from SOD1 aggregation data using Kaplan-Meier estimators.

Biochemical and functional characterization of OsCSD3, a novel CuZn superoxide dismutase from rice.

Superoxide dismutases (SODs, EC 1.15.1.1) belong to an important group of antioxidant metalloenzymes. Multiple SODs exist for scavenging of reactive oxygen species (ROS) in different cellular compartments to maintain an intricate ROS balance. The present study deals with molecular and biochemical characterization of CuZn SOD encoded by LOC_Os03g11960 (referred to as OsCSD3), which is the least studied among the four rice isozymes. The OsCSD3 showed higher similarity to peroxisomal SODs in plants. The OsCSD3 transcript was up-regulated in response to salinity, drought, and oxidative stress. Full-length cDNA encoding OsCSD3 was cloned and expressed in Escherichia coli and analyzed for spectral characteristics. UV (ultraviolet)-visible spectroscopic analysis showed evidences of d-d transitions, while circular dichroism analysis indicated high ß-sheet content in the protein. The OsCSD3 existed as homodimer (∼36 kDa) with both Cu2+ and Zn2+ metal cofactors and was substantially active over a wide pH range (7.0-10.8), with optimum pH of 9.0. The enzyme was sensitive to diethyldithiocarbamate but insensitive to sodium azide, which are the characteristics features of CuZn SODs. The enzyme also exhibited bicarbonate-dependent peroxidase activity. Unlike several other known CuZn SODs, OsCSD3 showed higher tolerance to hydrogen peroxide and thermal inactivation. Heterologous overexpression of OsCSD3 enhanced tolerance of E. coli sod double-knockout (ΔsodA ΔsodB) mutant and wild-type strain against methyl viologen-induced oxidative stress, indicating the in vivo function of this enzyme. The results show that the locus LOC_Os03g11960 of rice encodes a functional CuZn SOD with biochemical characteristics similar to the peroxisomal isozymes.

Cloning, purification and enzymatic characterization of recombinant human superoxide dismutase 1 (hSOD1) expressed in Escherichia coli.

Superoxide dismutase 1 (SOD1) is a metalloenzyme that catalyzes the disproportionation of superoxide into molecular oxygen and hydrogen peroxide. In this study, the human SOD1 (hSOD1) gene was cloned, expressed and purified. The hSOD1 gene was amplified from a pool of Bxpc3 cell cDNAs by PCR and cloned into expression vector pET-28a (+). The recombinant soluble hSOD1 was expressed in E. coli BL21 (DE3) at 37°C and purified using nickel column affinity chromatography. Soluble hSOD1 was produced with a yield of 5.9 µg/mL medium. As metal ions can have a certain influence on protein structure and activity, we researched the influences of different concentrations of Cu2+ and Zn2+ on hSOD1 activity at induction and the time of activity detection. The results implied that Cu2+ and Zn2+ do not enhance SOD1 expression and solubility; they can, however, improve the catalytic activity at induction. Meanwhile, Cu2+ and Zn2+ also enhanced the enzyme activity at the time of detection. Furthermore, most other bivalent cations had the potential to replace Zn2+ and Cu2+, and also improved enzyme activity at the time of detection.

Selective binding, magnetic separation and purification of histidine-tagged protein using biopolymer magnetic core-shell nanoparticles.

In previous studies, we synthesized the magnetic core-shell structured Fe3O4/PMG/IDA-Ni2+ nanoparticles. The Ni2+ on the surface of nanoparticles provides abundant docking sites for histidine, and the composite nanoparticles showed potential applications in the separation and purification of histidine-tagged (His-tagged) proteins. Meanwhile, the presence of the superparamagnetic core (Fe3O4) in the nanoparticles allows them to be quickly separated and purified by an external magnetic field. Herein, the ability of magnetic nanoparticles to purify His-tagged human superoxide dismutase 1 (hSOD1) was verified. SDS-PAGE and activity data showed His-tagged hSOD1 specifically bound to Fe3O4/PMG/IDA-Ni2+, and there was no significant competition for binding between final and three intermediate products. The binding capacity of nanoparticles can reach to 62.0 mg/g (dry weight of hSOD1/nanoparticles). The nanoparticle-bound hSOD1 exhibited better thermal and storage stability compared to free hSOD1. Furthermore, the purification efficiency of the magnetic nanoparticles in the separation and purification of His-tagged proteins was comparable to the other two commercial materials (High Affinity Ni-NTA Resin, HisPur Ni-NTA Magnetic Beads). Finally, the magnetic nanoparticles can be reused in the binding of His-tagged protein for multiple times. In conclusion, the nanoparticles are ready to be applied in the separation and purification of His-tagged protein.

Partial biochemical characterization of Cu,Zn-superoxide dismutase extracted from eggs of hens (Gallus gallus domesticus).

Biochemical characteristics of Cu,Zn-SOD derived from hen egg white and egg yolk were determined, and compared with those of enzymes from erythrocytes of hens and SOD standard. The presence of dimer with a molecular weight of 33.38±0.34kDa, and pI of 6.30±0.15 was confirmed in samples of SOD extracted from egg yolk. Cu,Zn-SOD isolated from egg yolk had an optimum at pH 6. Average SOD activity in egg yolk was 98.5±19.5U·g-1 while in egg white reached 6.1±0.8U·g-1. Changes in SOD activity of the egg yolk during its storage for 200days were also described. FTIR analysis confirmed that the enzymatic protein described in this study was SOD, while MALDI-TOF analysis confirmed only SOD from erythrocytes. Since eggs are a cheap and easily obtainable source of SOD, this enzymatic protein could be used in food, cosmetic or pharmaceutical industries.