TY - JOUR
T1 - RIEDL tag
T2 - A novel pentapeptide tagging system for transmembrane protein purification
AU - Asano, Teizo
AU - Kaneko, Mika K.
AU - Kato, Yukinari
N1 - Funding Information:
This research was supported in part by AMED [grant numbers: JP20am0401013 , JP20am0101078 , JP20ae0101028 ] (Y·K.).
Funding Information:
Synthesized DNA (Eurofins Genomics KK, Tokyo, Japan) encoding wPDPN (accession No.: XM_007104824.2 ) plus an N-terminal RIEDL tag, which is recognized by an anti-RIEDL tag mAb (LpMab-7), was subcloned into a pCAG-Neo vector (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) using the In-Fusion HD Cloning Kit (Takara Bio, Inc., Shiga, Japan). This plasmid was named pCAG-Neo/RIEDL-wPDPN. DNA encoding the CD20 gene (IRAL012D02) was provided by the RIKEN BRC through the National BioResource Project of MEXT, Japan. The open reading frame of CD20 plus the inserted RIEDL tag between Pro169 and Ala170 of human CD20 was subcloned into a pCAG-Ble vector using In-Fusion HD Cloning Kit. This plasmid was named pCAG-Ble/CD20- 169 RIEDL 170 .
Publisher Copyright:
© 2020 The Authors
PY - 2020/9
Y1 - 2020/9
N2 - Affinity tag systems are an essential tool in biochemistry, biophysics, and molecular biology. Although several different tag systems have been developed, the epitope tag system, composed of a polypeptide “tag” and an anti-tag antibody, is especially useful for protein purification. However, almost all tag sequences, such as the FLAG tag, are added to the N- or C-termini of target proteins, as tags inserted in loops tend to disrupt the functional structure of multi-pass transmembrane proteins. In this study, we developed a novel “RIEDL tag system,” which is composed of a peptide with only five amino acids (RIEDL) and an anti-RIEDL monoclonal antibody (mAb), LpMab-7. To investigate whether the RIEDL tag system is applicable for protein purification, we conducted the purification of two kinds of RIEDL-tagged proteins using affinity column chromatography: whale podoplanin (wPDPN) with an N-terminal RIEDL tag (RIEDL-wPDPN) and human CD20 with an internal RIEDL tag insertion (CD20-169RIEDL170). Using an LpMab-7-Sepharose column, RIEDL-wPDPN and CD20-169RIEDL170 were efficiently purified in one-step purification procedures, and were strongly detected by LpMab-7 using Western blot and flow cytometry. These results show that the RIEDL tag system can be useful for the detection and one-step purification of membrane proteins when inserted at either the N-terminus or inserted in an internal loop structure of multi-pass transmembrane proteins.
AB - Affinity tag systems are an essential tool in biochemistry, biophysics, and molecular biology. Although several different tag systems have been developed, the epitope tag system, composed of a polypeptide “tag” and an anti-tag antibody, is especially useful for protein purification. However, almost all tag sequences, such as the FLAG tag, are added to the N- or C-termini of target proteins, as tags inserted in loops tend to disrupt the functional structure of multi-pass transmembrane proteins. In this study, we developed a novel “RIEDL tag system,” which is composed of a peptide with only five amino acids (RIEDL) and an anti-RIEDL monoclonal antibody (mAb), LpMab-7. To investigate whether the RIEDL tag system is applicable for protein purification, we conducted the purification of two kinds of RIEDL-tagged proteins using affinity column chromatography: whale podoplanin (wPDPN) with an N-terminal RIEDL tag (RIEDL-wPDPN) and human CD20 with an internal RIEDL tag insertion (CD20-169RIEDL170). Using an LpMab-7-Sepharose column, RIEDL-wPDPN and CD20-169RIEDL170 were efficiently purified in one-step purification procedures, and were strongly detected by LpMab-7 using Western blot and flow cytometry. These results show that the RIEDL tag system can be useful for the detection and one-step purification of membrane proteins when inserted at either the N-terminus or inserted in an internal loop structure of multi-pass transmembrane proteins.
KW - CD20
KW - Monoclonal antibody
KW - Peptide tag
KW - Podoplanin
KW - Protein purification
KW - RIEDL tag
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U2 - 10.1016/j.bbrep.2020.100780
DO - 10.1016/j.bbrep.2020.100780
M3 - Article
AN - SCOPUS:85087938759
VL - 23
JO - Biochemistry and Biophysics Reports
JF - Biochemistry and Biophysics Reports
SN - 2405-5808
M1 - 100780
ER -