Novel Selenium-containing Human Single-chain Variable Fragment with Glutathione Peroxidase Activity from Computer-aided Molecular Design

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.

Imprinted Human Serum Albumin with Antioxidant Activity

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.