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

Abstract

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.

Development of a Novel Anti−CD44 Monoclonal Antibody for Multiple Applications against Esophageal Squamous Cell Carcinomas

“…CD44v3−10 ectodomain (CD44ec)−secreting LN229 (LN229/CD44ec) was established by transfecting pCAG−Neo/PA−CD44ec−RAP−MAP into LN229 cells using the Neon transfection system. The amino acid sequences of the tag system in this study were as follows: PA tag [ 52 , 53 , 54 ], 12 amino acids (GVAMPGAEDDVV); RAP tag [ 55 , 56 ], 12 amino acids (DMVNPGLEDRIE); and MAP tag [ 57 , 58 ], 12 amino acids (GDGMVPPGIEDK).…”

“…After LN229/CD44ec was cultured using DMEM complete medium without G418, CD44ec was purified from the supernatants using RAP tag system, comprised of an anti−RAP tag mAb (clone PMab−2) and a RAP peptide (GDDMVNPGLEDRIE) [ 55 , 56 ]. The filtered culture supernatant (5 L) was passed through PMab−2−Sepharose (2 mL bed volume), and the same process was repeated three times.…”

Development of a Novel Anti-CD44 Monoclonal Antibody C44Mab-46 for Multiple Applications against Esophageal Squamous Cell Carcinomas

“…CD44v3-10 ectodomain (CD44ec)-secreting LN229 (LN229/CD44ec) was established by transfecting pCAG-Neo/PA-CD44ec-RAP-MAP into LN229 cells using the Neon transfection system. The amino acid sequences of the tag system in this study were as follows: PA tag [23][24][25], 12 amino acids (GVAMPGAEDDVV); RAP tag [26,27], 12 amino acids (DMVNPGLEDRIE); and MAP tag [28,29], 12 amino acids (GDGMVPPGIEDK).…”

“…After LN229/CD44ec was cultured using DMEM complete medium without G418, CD44ec was purified from the supernatants using RAP tag system, comprised an anti-RAP tag mAb (clone PMab-2) and a RAP peptide (GDDMVNPGLEDRIE) [26,27]. The filtered culture supernatant (5L) was passed through PMab-2-Sepharose (2 mL bed volume), and the same process was repeated three times.…”

Epitope Mapping of Anti-mouse CCR3 Monoclonal Antibodies using Flow Cytometry

“…: NM_007720.2) [32][33][34] were subcloned into a pCAG-Ble vector (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), respectively. Chimeric mutants mCCR8 (mCCR3p1-38), mCCR8 (mCCR3p96-111), mCCR8 (mCCR3p176-207), and mCCR8 (mCCR3p269-285) were produced with a RAP [40,41] and a MAP tag [42,43] at their Cterminus using the HotStar HiFidelity polymerase kit (Qiagen Inc., Hilden, Germany). Alanine (glycin) substitutions in the mCCR3 N-terminal region were conducted using QuikChange Lightning Site-Directed Mutagenesis Kits (Agilent Technologies Inc., Santa Clara, CA, USA).…”