Optimization and Identification of Prokaryotic Expression Conditions of PD0721 Single-Chain Antibody in vitro

Abstract:

This work aimed to construct the Escherichia coli expression system, obtain the optimum expression conditions and establish a robust purification method of single-chain antibody PD0721 against epidermal growth factor receptor variant type Ⅲ(EGFRvⅢ). The recombinant plasmid PD0721-pET-22(+) was constructed and transformed into Escherichia coli BL 21.The optimum concentration of isopropyl-β-D-thiogalactoside (IPTG), induction temperature,induction time and cell concentration (OD₆₀₀) of the PD0721 expression were investigated by SDS-PAGE method. Ni²⁺ affinity chromatography was employed to purify PD0721 protein. Western Blotting and N-terminal sequencing were used to identify PD0721 protein. Colony PCR and agarose gel electrophoresis confirmed the successful construction of PD0721-pET-22b(+) recombinant plasmid and PD0721 recombinant Escherichia coli BL 21. The optimum IPTG concentration, temperature, induction time and OD₆₀₀ of PD0721 expression were 0.6 μmol/L, 15 ℃, 12 h and 0.6. PD0721 protein of high purity could be obtained when the concentration of imidazole was 150 mmol/L during the Ni²⁺ affinity purification. SDS-PAGE and Western Blotting results showed that the molecular weight of the recombinant target protein was about 31 000, consistent with the theoretical molecular weight. N-terminal sequencing of the protein was also consistent with the theoretical sequence of PD0721. In summary, the in vitro prokaryotic expression system of anti-EGFRvⅢ antibody PD0721 was successfully constructed. The optimum expression conditions were obtained, and a feasible method for purifying PD0721 protein was established.