Purification and characterization of two PR-10 protein isoforms from the crude drug of Angelica sinensis / 生物工程学报

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.

Expression, purification, stability and transduction efficiency of full-length SOD2 recombinant proteins / 生物工程学报

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.

Expression, purification, stability and transduction efficiency of GST-SOD1-R9 fusion protein / 生物工程学报

The fusion of cell permeable peptide TAT and bifunctional antioxidant enzymes, GST (Glutathione sulfur transferase)-TAT-SOD1 (Cu, Zn superoxide dismutase), is an intracellular superoxide scavenger. Compared with SOD1-TAT, GST-TAT-SOD1 has better protective effect on oxidative damage but less transduction efficiency. A novel cell permeable bifunctional antioxidant enzymes with the fusion of GST, SOD1 and polyarginine R9 was constructed for higher transduction efficiency. The full nucleotide sequence of SOD1-R9 was synthesized and inserted into the prokaryotic expression vector pGEX-4T-1 with the GST tag. After the successful construction of the prokaryotic expression vectors of GST-SOD1-R9, the recombinant vector was then transformed into Escherichia coli BL21 (DE3) and the GST-SOD1-R9 fusion protein was produced with the induction of IPTG. The soluble expression of GST-SOD1-R9 fusion protein was combining with the induction temperature and time. The best soluble expression was obtained with the induction temperature of 25 ℃ and the induction time of 11 h. The fusion protein was purified through the combination of 80% ammonium sulfate precipitation and affinity chromatography using glutathione agarose, and verified by SDS-PAGE and special enzymatic activity. The thermal and pH stability of GST-SOD1-R9 fusion protein were analyzed and the SOD and GST activity of fusion protein were proved to be well maintained under physiological conditions. Finally, the transduction efficiency of GST-SOD1-R9 fusion protein was proved to be better than GST-TAT-SOD1 fusion protein (P<0.05). These works establish a foundation for further study of the protective effect of GST-SOD1-R9 fusion protein against oxidative damage.

Efficacy of topical application of PTD-SOD on wound healing in mice with mechanical injury / 中华创伤杂志

Objective To study the role of the oxidative stress in the development of wound healing and observe the effect of the antioxidant PTD-SOD on damage and inflammation reaction after mechanical wound. Methods In this experiment,acute wound healing model by removal the whole layer dorsal skin of the mice was prepared,SOD(3 000 U and 6 000 U)and the fusion protein PTD-SOD with different concentrations(1 000 U,3 000 U,6 000 U and 10 000 U)were used to deal with the wounds continuously for 13 days.The mice were divided into different concentration SOD treatment group and PTD-SOD treatment group,model control group,physiological saline treatment group and compound iodine solution control group.The wound healing situation and healing percentage of the fight and left skin wounds of each mouse in every group was recorded every day.At day 14 after wound,the wound healing skin of each group was removed and some were used to make 10%tissues homogenate for detecting the activities of superoxide dismutase(SOD),catalase(CAT),glutathione peroxidase(GSH-Px)and contents of malondialdehyde(MDA)and hydroxyproline(Hyp);in the meantime,the other removed skin were fixed in 10% formalin for observing the histopathological changes of the tissues. Results Compared with the model control group,the physiological saline treatment group and the compound iodine solution control group,the skin wound healing percentage was significantly(P<0.05 or P<0.01)improved,with increase of the activities of SOD,CAT,GSH-Px and contents of Hyp (P<0.05 or P<0.01)and decrease of MDA(P<0.05 or P<0.01) in the SOD groups or PTD-SOD groups (except for 10 000 U PTD-SOD group).When compared with the physiological saline treatment group or the compound iodine solution treatment group,the effect was similar to the model control group.In comparison to the SOD groups,under the same dosage and environment condition,the PTD-SOD groups were much better than SOD groups with regard to promoting skin wound healing percentage,increasing activities of antioxidases and contents of Hyp,decreasing contents of MDA.Among the PTD-SOD groups,the effect of high dosage 10 000 U on promoting skin wound healing was declined. Conclusions The oxidative stress may playan important role in the development of wound healing.Proper application of treatment with antioxidants is a alternative strategy in the early stage of wound.PTD-SOD is able to prevent the oxidative stress damage,inhibit inflammatory infiltration and promote skin wound healing efficiently.

PTD mediated protein transduction technology and its application in medical field / 生物医学工程学杂志

The delivery of bioactive macromolecular substances into cells provides an efficient approach to changing cellular conditions, and is thus of enormously potential therapeutic significance. It has also been an extremely difficult approach due the the impediment and protective nature of cell membrance until the protein transduction domain's (PTD's) capability to ferry macromolecule across cell membrance was discovered. PTD's efficient transductive function has rendered an exciting promise to the clinical treatment of diseases, therapeutic proteins drug development, and basic medical and applied research. The technology has been successfully applied to deliver a variety of substances into cells or tissue organs, and its superior application values have been explicitly demonstrated.

Expression in Escherichia coli, purification and enzymatic properties of chicken aminopeptidase H / 生物工程学报

Aminopeptidase H (APH) is an universally distributed aminoendopeptidase in the tissue of many organism. However, it is hard to investigate its mechanism underlying the catalysis and the function in cell. In this paper, the full DNA sequence of this enzyme was cloned from chicken liver, then subcloned to the vector pET22 b(+). The recombined vector was transformed into E. coli Rosetta(DE3), and the APH gene was expressed by the induction of IPTG. It was found the recombinant protein exhibited same mo lecular weight as authentic APH on SDS-PAGE analysis; the expression level increased with induction time and approached maximum of 94.7 mg/L till 6 hours, which contained 16.7% of the total protein. Moreover, this recombinant protein showed similar prop erties of subunit composition, thermal stability and optimum pH with native APH, based on the enzymatic assay, purification and analysis of enzymological properties. Therefore, it is confirmed that APH was expressed in this prokaryote system with a high-level of 1636 u/L aminopeptidase activity. These results would help to elucidate the catalysis mechanism and biological function of APH by providing enough material.

A study of the blood supply relevant to prevention of nipple-areola necrosis in female breast operation / 中国普通外科杂志

Objective To study the arterial blood supply of nipple-areola and provide the anatomical basis for avoiding nipple-areola necrosis in breast operation. Methods The vascular structure of nipple-areola of 26 female breasts in 13 cadavers were studied. Results The nipple-areola mainly accepted arterial blood supply from branches of the lateral thoracic artery and the internal thoracic artery. The 2nd~4th intercostal ~perforating branches of the internal thoracic artery and branches of the lateral thoracic artery reach the base of nipple-areola from a superior,medial and upper lateral direction by passing between lobules of mammary gland, then ascend between the lacteal ducts to supply the nipple-areola; the perforators of the lateral thoracic artery and the superticial breast perforators of internal thoracic artery, formed extensive anastomoses ~subcutaneously , and particulatly under areola formed arterial rete, from which branches were given out to ~nipple-areola . The intercostal perforators and thoracoacromial perforators did not supply the nipple-areola. Conclusions When nipple-sparing mastectomy is performed, in order to avoid nipple-areola necrosis,it is necessary to protect the arterial rete under the areola, and thus, the thickness of areolar skin flap should not be less than 0.5cm; to ensure the blood supply of nipple-areola from the internal thoracic artery and the ~lateral thoracic artery in breast reduction, the superior-medial or superior-lateral breast pedicle should be used and the thickness of preserved posterior breast should not be less than 1.5cm.