Development of RAP Tag, a Novel Tagging System for Protein Detection and Purification

Fujii, Yuki; Kaneko, Mika K.; Ogasawara, Satoshi; Yamada, Shinji; Yanaka, Miyuki; Nakamura, Takuro; Saidoh, Noriko; Yoshida, Kanae; Honma, Ryusuke; Kato, Yukinari

doi:10.1089/mab.2016.0052

Cited by 42 publications

(37 citation statements)

References 35 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…SIZ1-FLAG or SIZ1(C162S)-FLAG was immunoprecipitated with anti-DYKDDDK antibody. The immunoprecipitant was detected with anti-RAP tag antibody 45 . These results suggest that SIZ1 interacts with ATX1 in vivo.…”

Section: Resultsmentioning

confidence: 99%

“…The PCR products containing His-tag (HHHHHH), and FLAG tag (DYKDDDDK) were produced with the primers pBYR2HS-Flag-F, FLAG-His, and pBYR2HS-FlagHis-R (Supplementary Table 1). The PCR products containing His-tag (HHHHHH), RAP tag (DMVNPGLEDRIE) 45 , and the recognition site for HRV 3 C protease (LEVLFQGP), were produced with the primers pBYR2HS-HRV3C-F, HRV3C-RAP-His, and pBYR2HS-stopHis-R (Supplementary Table 1). These PCR products were introduced into the SalI-digested pBYR2HS with an In-Fusion HD Cloning Fig.…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

The PHD finger of Arabidopsis SIZ1 recognizes trimethylated histone H3K4 mediating SIZ1 function and abiotic stress response

2020

View full text Add to dashboard Cite

Arabidopsis SIZ1 encodes a SUMO E3 ligase to regulate abiotic and biotic stress responses. Among SIZ1 or mammalian PIAS orthologs, plant SIZ1 proteins contain the plant homeodomain (PHD) finger, a C 4 HC 3 zinc finger. Here, we investigated the importance of PHD of Arabidopsis SIZ1. The Pro SIZ1 ::SIZ1(ΔPHD):GFP was unable to complement growth retardation, ABA hypersensitivity, and the cold-sensitive phenotype of the siz1 mutant, but Pro SIZ1 ::SIZ1: GFP could. Substitution of C162S in the PHD finger was unable to complement the siz1 mutation. Tri-methylated histone H3K4 (H3K4me3) was recognized by PHD, not by PHD (C162S). WRKY70 was up-regulated in the siz1-2 mutant and H3K4me3 accumulated at high levels in the WRKY70 promoter. PHD interacts with ATX, which mediates methylation of histone, probably leading to suppression of ATX's function. These results suggest that the PHD finger of SIZ1 is important for recognition of the histone code and is required for SIZ1 function and transcriptional suppression.

show abstract

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

The PHD finger of Arabidopsis SIZ1 recognizes trimethylated histone H3K4 mediating SIZ1 function and abiotic stress response

2020

View full text Add to dashboard Cite

show abstract

“…Anti-EGFR hybridomas were produced, as previously mentioned (15). The ectodomain of EGFR with N-terminal PA tag (16), C-terminal RAP tag (17), and MAP tag (14) (EGFRec) was purified from the supernatant of LN229/EGFRec using the anti-RAP tag previously described (17).…”

Section: Methodsmentioning

confidence: 99%

A novel anti‑EGFR monoclonal antibody (EMab‑17) exerts antitumor activity against oral squamous cell carcinomas via antibody‑dependent cellular cytotoxicity and complement‑dependent cytotoxicity

et al. 2020

Self Cite

View full text Add to dashboard Cite

The epidermal growth factor receptor (EGFR) is a member of the human epidermal growth factor receptor (HER) family of receptor tyrosine kinases; it is a transmembrane receptor involved in cell growth and differentiation. EGFR homodimers or heterodimers in combination with other HER members, such as HER2 and HER3, activate downstream signaling cascades in many types of cancer, including oral squamous cell carcinoma (OSCC). The present study produced novel anti-EGFR monoclonal antibodies (mAbs) possessing antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), and investigated antitumor activity. Mice were immunized with an EGFR-overexpressed glioblastoma cell line, LN229 (LN229/EGFR), after which ELISA was performed using recombinant EGFR. mAbs were subsequently selected according to their efficacy for LN229/EGFR, as determined via flow cytometry. After determining the subclass of mAbs, the EMab-17 (IgG 2a , kappa) clone exhibited ADCC and CDC activities against two OSCC cell lines, HSC-2 and SAS. Furthermore, EMab-17 exerted antitumor activities against mouse xenograft models using HSC-2 and SAS, indicating that EMab-17 may be used in an antibody-based therapy for EGFR-expressing OSCC.

show abstract

“…We previously inserted dPDPN with an N-terminal PA tag (GVAMPGAEDDVV) (19) and a C-terminal RAP tag (DMVNPGLEDRIE) (20) -MAP tag (GDGMVPPGIEDK) (21) (PA-dPDPN-RAP-MAP) in a pCAG-Ble vector (FUJIFILM Wako Pure Chemical Corporation). (11) CHO-K1 cells (American Type Culture Collection, Manassas, VA) were transfected with pCAG-Ble/PA-dPDPN-RAP-MAP using Gene Pulser Xcell electroporation system (Bio-Rad Laboratories, Inc., Berkeley, CA) for developing CHO/dPDPN.…”

Section: Cell Lines and Animalsmentioning

confidence: 99%

Antibody–Drug Conjugates Using Mouse–Canine Chimeric Anti-Dog Podoplanin Antibody Exerts Antitumor Activity in a Mouse Xenograft Model

Kato

Ito

Ohishi

et al. 2020

Monoclonal Antibodies in Immunodiagnosis and Immunotherapy

Self Cite

View full text Add to dashboard Cite

Antibody-drug conjugates (ADCs), which consist of a monoclonal antibody (mAb), a linker, and a payload, can deliver a drug to cancer tissues. We previously produced an anti-dog podoplanin (dPDPN) mAb, PMab-38, which reacts with dPDPN-expressing canine melanomas and squamous cell carcinomas (SCCs), but not with dPDPN-expressing canine type I alveolar cells or lymphatic endothelial cells, indicating that PMab-38 possesses cancer specificity. In this study, we developed an ADC, P38B-DM1, using the mouse-canine chimeric anti-dPDPN antibody, P38B as the antibody, a peptide linker, and emtansine as the payload using the chemical conjugation by affinity peptide (CCAP) method. We investigated its cytotoxicity against dPDPN-overexpressed Chinese hamster ovary (CHO/dPDPN) cells in vitro and its antitumor activity using a mouse xenograft model of CHO/dPDPN cells. P38B-DM1 showed cytotoxicity to CHO/dPDPN cells in a dose-dependent manner in vitro. Furthermore, P38B-DM1 exhibited higher antitumor activity than P38B in the mouse xenograft model. These results suggest that P38B-DM1, developed using the CCAP method, is useful for antibody therapy against dPDPN-expressing canine SCCs and melanomas.

show abstract

Development of RAP Tag, a Novel Tagging System for Protein Detection and Purification

Cited by 42 publications

References 35 publications

The PHD finger of Arabidopsis SIZ1 recognizes trimethylated histone H3K4 mediating SIZ1 function and abiotic stress response

The PHD finger of Arabidopsis SIZ1 recognizes trimethylated histone H3K4 mediating SIZ1 function and abiotic stress response

A novel anti‑EGFR monoclonal antibody (EMab‑17) exerts antitumor activity against oral squamous cell carcinomas via antibody‑dependent cellular cytotoxicity and complement‑dependent cytotoxicity

Antibody–Drug Conjugates Using Mouse–Canine Chimeric Anti-Dog Podoplanin Antibody Exerts Antitumor Activity in a Mouse Xenograft Model

Contact Info

Product

Resources

About