378242 The Introduction of PMMA-Tag to VHH for Improving Recovery and Immobilization Rate of Vhhs

Tuesday, November 18, 2014: 8:50 AM
312 (Hilton Atlanta)
Bongmun Kang1, Kagenari Yamakawa2, Michimasa Kishimoto2 and Yoichi Kumada2, (1)Biomolecular Engineering, Kyoto Insititute of Technology, Kyoto, Japan, (2)Bio-molecular Engineering, Kyoto Institute of Technology, Kyoto, Japan

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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See more of this Session: AIChE-SCEJ Joint Session: Bioseparations and Bionanotechnology
See more of this Group/Topical: Separations Division