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

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. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1563–1570, 2015