Lablog4-50:Efficient preparation and site-directed immobilization of VHH antibodies by genetic fusion of poly(methylmethacrylate)-binding peptide (PMMA-Tag)

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Yoichi Kumada, Bongmun Kang, Kagenari Yamakawa, Michimasa Kishimoto, Jun-ichi Horiuchi

First author

Yoichi Kumada

Corresponding author

Yoichi Kumada

Publication Style

Journal name Biotechnology Progress


Volume, issue, pages

31(6) 1563-1570


A PMMA-binding peptide (PMMA-tag) was genetically fused with the C-terminal region of an anti-human chorionic gonadotropin (hCG) single-domain antibody (VHH). It was over-expressed in an insoluble fraction of E. coli cells, and recovered in the presence of 8 M urea via one-step IMAC purification. Monomeric and denatured PMMA-tag-fused VHH (VHH-PM) was successfully prepared via the reduction and oxidation of VHH-PM at a concentration less than 1 mg/mL in the presence of 8 M of urea. Furthermore, the VHH-PM was refolded with a recovery of more than 95% by dialysis against 50 mM TAPS at pH 8.5, because the genetic fusion of PMMA-tag resulted in a decrease in the apparent isoelectric point (pI) of the fusion protein, and its solubility at weak alkaline pH was considerably increased. The antigen-binding activities of VHH-PM in the adsorptive state were 10-fold higher than that of VHH without a PMMA-tag. The density of VHH-PM on a PMMA plate was twice that of VHH, indicating that the site-directed attachment of a PMMA-tag resulted in positive effects to the adsorption amount as well as to the orientation of VHH-PM in its adsorptive state. The preparation and immobilization methods for VHH-PM against hCG developed in the present study were further applied to VHH-PMs against four different antigens, and consequently, those antigens with the concentrations lower than 1 ng/mL were detected by the sandwich ELISA. Thus, the VHH-PMs developed in the present study are useful for preparation of high-performance and economical immunosorbent for detection of biomarkers.