(237b) The Introduction of PMMA-Tag to VHH for Improving Recovery and Immobilization Rate of Vhhs

Kang, B., Kyoto Insititute of Technology
Yamakawa, K., Kyoto Institute of Technology
Kishimoto, M., Kyoto Institute of Technology
Kumada, Y., Kyoto Institute of Technology

The PMMA-tag was genetically fused with the C-terminal region of VHH molecules.  This antibody, VHH, is known as a single domain antibody, which is devoid of light chains.  Genetic fusion of negatively-charged poly(methyl methacrylate)-binding peptide (PMMA-tag) to its C-terminus, resulted in decrease in apparent isoelectric point (pI) of VHH, and consequently, PMMA-tag-fused VHH (VHH-PM) could be refolded with more than 95% recovery by dialysis against 50 mM TAPS at pH 8.5. Adsorption ability of PMMA-tag was remained even after refolding of VHH-PM. According to the results of sandwich ELISA that VHH and VHH-PM were immobilized onto the surface of PMMA plate, antigen-binding activity of VHH-PM in adsorption state was 8-fold higher than that of VHH without PMMA-tag. Thus, PMMA-tag developed in this study will be significantly useful for refolding and site-specific immobilization of VHHs with broader species.


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