In order to enhance the glutathione peroxidase（GPX） catalytic activity of the selenium-containing single-chain variable fragments（Se-scFv）, a novel human scFv was designed on the basis of the structure of human an...In order to enhance the glutathione peroxidase（GPX） catalytic activity of the selenium-containing single-chain variable fragments（Se-scFv）, a novel human scFv was designed on the basis of the structure of human antibody and optimized via bioinformatics methods such as homologous sequence analysis, three-dimensional（3D） model building, binding-site analysis and docking. The DNA sequence of the new human scFv was synthesized and cloned into the expression vector pET22b（＋）, then the scFv protein was expressed in soluble form in Escherichia coli BL21（DE3） and purified by Ni2＋-immobilized metal affinity chromatography（IMAC）. The serine residue of scFv in the active site was converted into selenocysteine（Sec） with the chemical modification method, thus, the human Se-scFv with GPX activity was obtained. The GPX activity of the Se-scFv protein was characterized. Compared with other Se-scFv, the new human Se-scFv showed similar efficiency for catalyzing the reduction of hydrogen peroxide by glutathione. It exhibited pH and temperature dependent catalytic activity and a typical ping-pong kinetic mechanism.展开更多
In order to create a new mimic of glutathione peroxidase（GPx）, bioimprinting was used to generate gluta-thione（GSH） binding site and chemical modification was used to incorporate catalytic group selenocystine（Sec...In order to create a new mimic of glutathione peroxidase（GPx）, bioimprinting was used to generate gluta-thione（GSH） binding site and chemical modification was used to incorporate catalytic group selenocystine（Sec）. Human serum albumin（HSA） and S-substituted dinitrophenyl glutathione（GSH-S-DNP） were chosen as the imprinted matrix and imprinting template, respectively, to generate a GSH-imprinted protein（GSH-HSA） by bioimprinting. Sec was incorporated into the GSH-HSA by chemical modification to give a new GPx mimic（Se-GSH-HSA）. Se-GSH-HSA displayed considerably higher GPx activity than non-printed HSA（Se-HSA）. The enzymic properties and kinetics of Se-GSH-HSA were studied. Moreover, Se-GSH-HSA was confirmed to have stronger antioxidant ability to protect mitochondria against oxidative damage with ferrous sulfate/ascorbate-induced mitochondria damage model, indicating that Se-GSH-HSA has potential application in medicine.展开更多
基金Supported by the National Natural Science Foundation of China(No.30970608)the Applicative Technological Project of Bureau of Science and Technology of Changchun City, China(No.2009045)+1 种基金the Development and Planning Major Program of Jilin Provincial Science and Technology Department, China(No.20100948)the Innovation Method Fund of China (No.2008IM040800)
文摘In order to enhance the glutathione peroxidase（GPX） catalytic activity of the selenium-containing single-chain variable fragments（Se-scFv）, a novel human scFv was designed on the basis of the structure of human antibody and optimized via bioinformatics methods such as homologous sequence analysis, three-dimensional（3D） model building, binding-site analysis and docking. The DNA sequence of the new human scFv was synthesized and cloned into the expression vector pET22b（＋）, then the scFv protein was expressed in soluble form in Escherichia coli BL21（DE3） and purified by Ni2＋-immobilized metal affinity chromatography（IMAC）. The serine residue of scFv in the active site was converted into selenocysteine（Sec） with the chemical modification method, thus, the human Se-scFv with GPX activity was obtained. The GPX activity of the Se-scFv protein was characterized. Compared with other Se-scFv, the new human Se-scFv showed similar efficiency for catalyzing the reduction of hydrogen peroxide by glutathione. It exhibited pH and temperature dependent catalytic activity and a typical ping-pong kinetic mechanism.
基金Supported by the National Basic Research Program of China(No. 2007CB714503)the National Natural Science Foundation of China(No.30970608)+5 种基金the Applicative Technological Project of Bureau of Science and Technology of Changchun CityChina (No.2009045)the Development and Planning Major Program of Jilin Provincial Science and Technology Department China (No.20100948)the Innovation Method Fund of China(No.2008IM040800)the Open Project of State Key Laboratory of Supramolecular Structure and Materials of China(No.SKLSSM200915)
文摘In order to create a new mimic of glutathione peroxidase（GPx）, bioimprinting was used to generate gluta-thione（GSH） binding site and chemical modification was used to incorporate catalytic group selenocystine（Sec）. Human serum albumin（HSA） and S-substituted dinitrophenyl glutathione（GSH-S-DNP） were chosen as the imprinted matrix and imprinting template, respectively, to generate a GSH-imprinted protein（GSH-HSA） by bioimprinting. Sec was incorporated into the GSH-HSA by chemical modification to give a new GPx mimic（Se-GSH-HSA）. Se-GSH-HSA displayed considerably higher GPx activity than non-printed HSA（Se-HSA）. The enzymic properties and kinetics of Se-GSH-HSA were studied. Moreover, Se-GSH-HSA was confirmed to have stronger antioxidant ability to protect mitochondria against oxidative damage with ferrous sulfate/ascorbate-induced mitochondria damage model, indicating that Se-GSH-HSA has potential application in medicine.
