1. [Studies on the optimal expression condition, purification and its characterization of ScFv-2F3].
- Author
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Luo YM, Mu Y, Wei JY, Yan GL, and Luo GM
- Subjects
- Antibodies, Catalytic chemistry, Antibodies, Catalytic genetics, Antibodies, Catalytic isolation & purification, Bioreactors microbiology, Cloning, Molecular, Escherichia coli, Immunoglobulin Fragments chemistry, Immunoglobulin Fragments genetics, Immunoglobulin Fragments isolation & purification, Inclusion Bodies metabolism, Protein Folding, Protein Renaturation, Recombinant Proteins biosynthesis, Recombinant Proteins chemistry, Recombinant Proteins genetics, Recombinant Proteins isolation & purification, Selenium metabolism, Antibodies, Catalytic biosynthesis, Gene Expression, Immunoglobulin Fragments biosynthesis
- Abstract
The expression vectors of the gene encoding ScFv-2F3 were transformed into E. coli BL21(DE3). Clones of higher expression were first selected, then were grown in the presence of IPTG at 37 degrees C to induce its expression. The culture conditions were carefully optimized. It was found that optimal conditions were as follows: the induction was started as OD590 reached to 1.0-1.8; the concentration of IPTG was 0.3-0.5 mmol/L and induction time is 7 h. The yield of ScFv-2F3 expressed in the selected clones is about 20% of the total proteins. The optimal culture conditions were successfully applied to fermenter of 50 L. The conditions of washing the inclusion bodies were also optimized. A two-step method was used to renature the inclusion body. The expression product of interest and its biological activities were characterized with Western blotting and ELISA. A novel selenium-containing single-chain abzyme with GPX activity was prepared.
- Published
- 2002