Construction of Fluorescent Immunosensor Quenchbody to Detect His-Tagged Recombinant Proteins Produced in Bioprocess
Xuerao Ning, Takanobu Yasuda, Tetsuya Kitaguchi, Hiroshi Ueda
Abstract
With the widespread application of recombinant DNA technology, many useful substances are produced by bioprocesses. For the monitoring of the recombinant protein production process, most of the existing technologies are those for the culture environment (pH, O2, etc.). However, the production status of the target protein can only be known after the subsequent separation and purification process. To speed up the monitoring of the production process and screening of the higher-yield target protein variants, here we developed an antibody-based His-tag sensor Quenchbody (Q-body), which can quickly detect the C-terminally His-tagged recombinant protein produced in the culture medium. Compared with single-chain Fv-based Q-body having one dye, the Fab-based Q-body having two dyes showed a higher response. In addition, not only was fluorescence response improved but also detection sensitivity by the mutations of tyrosine to tryptophan in the heavy chain CDR region. Moreover, the effect of the mutations on antigen-binding was successfully validated by molecular docking simulation by CDOCKER. Finally, the constructed Q-body was successfully applied to monitor the amount of anti-SARS CoV-2 nanobody secreted into the Brevibacillus culture media.