TY - JOUR
T1 - RAP Tag and PMab-2 Antibody
T2 - A Tagging System for Detecting and Purifying Proteins in Plant Cells
AU - Miura, Kenji
AU - Yoshida, Hideki
AU - Nosaki, Shohei
AU - Kaneko, Mika K.
AU - Kato, Yukinari
N1 - Publisher Copyright:
© Copyright © 2020 Miura, Yoshida, Nosaki, Kaneko and Kato.
PY - 2020/9/10
Y1 - 2020/9/10
N2 - 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.
AB - 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.
KW - RAP tag
KW - agroinfiltration
KW - monoclonal antibody
KW - plant biochemistry
KW - protein expression
KW - protein purification
KW - tagging system
KW - transient expression
UR - http://www.scopus.com/inward/record.url?scp=85091515881&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85091515881&partnerID=8YFLogxK
U2 - 10.3389/fpls.2020.510444
DO - 10.3389/fpls.2020.510444
M3 - Article
AN - SCOPUS:85091515881
VL - 11
JO - Frontiers in Plant Science
JF - Frontiers in Plant Science
SN - 1664-462X
M1 - 510444
ER -