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.
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.
