Generation of selenoprotein with glutathione peroxidase activity by chemical modification of the single-chain variable fragment expressed in a single-protein production system and its antioxidant ability
| Autor: | Gang Zhao, Ya-wei Xu, Yan Ganglin, Jun-jie Xu, Cheng Wang, Chun-Hong Sui, Ying Mu, Yang Zhao, Pingsheng Gong, Guimin Luo, Shao-wu Lü |
|---|---|
| Rok vydání: | 2013 |
| Předmět: |
chemistry.chemical_classification
GPX1 Antioxidant biology Selenocysteine Chemistry Glutathione peroxidase medicine.medical_treatment Biomedical Engineering chemical and pharmacologic phenomena Bioengineering Glutathione respiratory system Applied Microbiology and Biotechnology Molecular biology chemistry.chemical_compound Biochemistry biology.protein medicine Single-chain variable fragment Selenoprotein Biotechnology Peroxidase |
| Zdroj: | Biotechnology and Bioprocess Engineering. 18:27-34 |
| ISSN: | 1976-3816 1226-8372 |
| DOI: | 10.1007/s12257-012-0174-3 |
| Popis: | Glutathione peroxidase (GPX) is one of the important members of the antioxidant enzyme family. It can catalyze the reduction of hydroperoxides with glutathione to protect cells against oxidative damage. Single-chain variable fragment (scFv) can be converted into seleniumcontaining single-chain variable fragment (Se-scFv) by chemical modification of the hydroxyl groups in scFv, thus Se-scFv possesses GPX activity and becomes a prodrug. To improve the expression of scFv and simplify its purification steps, Single-protein production (SPP) system was used to express scFv and chemical modification was used to synthesize Se-scFv. Therefore, we must construct a new scFv-WCD1-lessACA gene, which can express its mRNA not containing any ACA sequences and express its amino acid sequence of target protein (scFv) being same to scFv-WCD1. In this way, the scFv-WCD1-lessACA can be only expressed in SPP system and no other background proteins in the cells could be expressed. The expression results showed that high level of scFv-WCD1-lessACA synthesis was at least sustained for 96 h in the virtual absence of background protein synthesis. Then, selenocysteine (Sec) was incorporated into the scFv-WCD1-lessACA by chemical modification and resulted in Se-scFv-WCD1-lessACA. The enzymatic characteristics of Se-scFv-WCD1-lessACA were determined. GPX activity was 2,563 U/μmol, its binding constant for GSH was 0.687 ×105/mol. Moreover, Se-scFv-WCD1-lessACA was confirmed to have a strong antioxidant ability to protect mitochondria against oxidative damage induced by Vc/Fe2+ (mitochondrial damage model), suggesting that Se-scFv-WCD1-lessACA has potential application for protection of mitochondrial damage induced by reactive oxygen species (ROS). |
| Databáze: | OpenAIRE |
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