RAP Tag and PMab-2 Antibody: A Tagging System for Detecting and Purifying Proteins in Plant Cells

• Main Authors: Kenji Miura, Hideki Yoshida, Shohei Nosaki, Mika K. Kaneko, Yukinari Kato
• Format: Article
• Language: English
• Published: Frontiers Media S.A. 2020-09-01
• Series: Frontiers in Plant Science
• Subjects: transient expression; tagging system; RAP tag; agroinfiltration; monoclonal antibody; protein expression
• Online Access: https://www.frontiersin.org/article/10.3389/fpls.2020.510444/full

An affinity tag system requires both high affinity and specificity. The RAP tag epitope DMVNPGLEDRIE, derived from rat podoplanin (PDPN), is specifically recognized by PMab-2 monoclonal antibodies in rats. Here, we demonstrated that high levels of PMab-2 can be produced in Nicotiana benthamiana and plant-derived PMab-2 possesses similar activity to CHO-derived PMab-2, and the RAP tag presents a useful tagging system for detecting and purifying proteins from plant cells. The heavy chain of PMab-2 fused with KDEL, an endoplasmic reticulum retention sequence, and the light chain of the antibody were introduced into N. benthamiana by agroinfiltration. The expression of PMab-2 peaked 4 days after agroinfiltration, and approximately 0.3 mg/g fresh weight of the antibody was accumulated. After purification, the plant-derived PMab-2 successfully recognized rat PDPN expressed in CHO-K1 cells and exhibited almost the same binding activity as CHO-derived PMab-2. The RAP-tagged proteins expressed in plant cells were specifically recognized by PMab-2. These results indicate that PMab-2 can accumulate at high levels in N. benthamiana and is easily purified and that the RAP tagging system presents a useful tool for detecting and purifying proteins of interest in plant cells.