Recombinant Expression, Renaturation and Biological Activity of Temperature-Responsive Nanobodies against Aflatoxin B1
2023
ZHANG Leping, TU Zhui, LI Yanping, LI Xiaojiang, SHUAI Wenyuan, ZHANG Hang, HE Qinghua
In this study, temperature-responsive anti-aflatoxin B1 (AFB1) nanoantibodies were obtained by fusion expression of elastin-like polypeptides (ELPs) of different lengths with nanoantibodies. The recombinant expression vectors pET30a-G8-ELP20, pET30a-G8-ELP40, pET30a-G8-ELP60 and pET30a-G8-ELP80 were synthesized by recursive directional ligation (RDL) and transformed into Escherichia coli BL21 (DE3) cells, separately. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed that the four fusion proteins were expressed in the form of inclusion bodies in the bacterial precipitate. The inclusion bodies were dissolved in denaturing buffer and refolded by dilution, inverse transition cycling (ITC) purification, dialysis or column refolding. SDS-PAGE analysis showed that the purity of the four proteins obtained achieved using the four refolding methods was similar. After refolding by ITC, the highest yield of 83%, 90.7%, 89.5%, and 88.3% was obtained for the four fusion proteins, respectively. The half maximal inhibitory concentration (IC50) of G8-ELP80 obtained by dilution refolding was the lowest (4.35 ng/mL), as determined by indirect competitive enzyme-linked immunosorbent assay (ELISA). The phase transition temperatures (Tt) of nanoantibodies tagged with the different ELPs, as determined by the turbidity method, were 45, 38, 32 and 28 ℃, respectively. Circular dichroism (CD) spectroscopy showed that the main secondary structures of the four fusion proteins were β-sheet and β-turn. This study provides a basis for subsequent AFB1 detection and analysis.
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