Superoxide dismutase (SOD) family is necessary to protect cells from the toxicity of reactive oxygen species produced during normal metabolism. Among SODs, manganese-containing superoxide dismutase (Mn-SOD, SOD2) is the most important one. The DNA fragment containing the full nucleotide of full-length human SOD2 was synthesized and inserted into the prokaryotic expression vector pGEX-4T-1 with tag GST. DNA construct was then transformed into Escherichia coli BL21 (DE3) and expression was induced with IPTG at 25 ℃. The recombinant fusion protein GST-SOD2 (46 kDa) was purified from the bacterial lysate by GST resin column affinity chromatography. GST tag was cleaved with thrombin, and a crude SOD2 recombinant protein (25 kDa) was obtained and further purified by heparin affinity chromatography. Activities of the two SOD2 proteins were 1 788 and 2 000 U/mg, respectively. Both SOD2 proteins were stable under physiological condition and cell-penetrating (P<0.05). Our findings open the possibility to study the structure and effects of two full-length recombinant SOD2 proteins.

Two proteins of similar molecular weight (named as ASPR-C-1 and ASPR-C-2) from the crude drug of Angelica sinensis were purified and characterized by 80% ammonium sulfate precipitation, Sephadex G-50 gel filtration chromatography, and DEAE-Sepharose anion exchange chromatography. The molecular weight of ASPR-C-1 and ASPR-C-2 on SDS-PAGE was 17.33 kDa and 17.18 kDa, respectively. They were mainly monomeric in solution, but partially formed dimers and they were glycoproteins with glycosyl content of 2.6% and 8.2%, respectively. Both ASPR-C-1 and ASPR-C-2 were identified to be members of pathogenesis-related 10 family of proteins by matrix-assisted laser desorption ionization time-of-flight mass spectrometry and have ribonuclease activities with the specific activity of 73.60 U/mg and 146.76 U/mg, respectively. The optimum pH of the two isoforms was similar, at about 5.6, while their optimum temperatures were different. The optimum temperature of ASPR-C-1 was 50 ℃, and that of ASPR-C-2 was 60 ℃. Both isoforms presented highest thermal stability at 60 ℃. However, ASPR-C-2 was more thermotolerant than ASPR-C-1. The latter was rapidly inactivated and retained only about 20% residual activity while the former still maintained about 80% of its original activity at a higher treatment temperature (80 to 100 ℃). In addition, Fe²⁺ had an activating effect on the ribonuclease activities of two isoforms while Ca²⁺, Mg²⁺, Zn²⁺, Mn²⁺, Ag⁺, Cu²⁺, EDTA (Elhylene diamine tetraacetic acid), dithiothreitol and sodium dodecylsulphate showed different degrees of inhibition of the enzyme activities. Our findings provide a foundation for further research on the biological function of PR-10 protein from Angelica sinensis.
Angelica sinensis ; Chromatography, Gel ; Chromatography, Ion Exchange ; Electrophoresis, Polyacrylamide Gel ; Enzyme Stability ; Hydrogen-Ion Concentration ; Kinetics ; Molecular Weight ; Protein Isoforms ; Temperature