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RIEDL tag: A novel pentapeptide tagging system for transmembrane protein purification

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...

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Autores principales: Asano, Teizo, Kaneko, Mika K., Kato, Yukinari
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Elsevier 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7369347/
https://www.ncbi.nlm.nih.gov/pubmed/32715101
http://dx.doi.org/10.1016/j.bbrep.2020.100780
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author Asano, Teizo
Kaneko, Mika K.
Kato, Yukinari
author_facet Asano, Teizo
Kaneko, Mika K.
Kato, Yukinari
author_sort Asano, Teizo
collection PubMed
description 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-(169)RIEDL(170)). Using an LpMab-7-Sepharose column, RIEDL-wPDPN and CD20-(169)RIEDL(170) 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.
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spelling pubmed-73693472020-07-23 RIEDL tag: A novel pentapeptide tagging system for transmembrane protein purification Asano, Teizo Kaneko, Mika K. Kato, Yukinari Biochem Biophys Rep Research Article 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-(169)RIEDL(170)). Using an LpMab-7-Sepharose column, RIEDL-wPDPN and CD20-(169)RIEDL(170) 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. Elsevier 2020-07-17 /pmc/articles/PMC7369347/ /pubmed/32715101 http://dx.doi.org/10.1016/j.bbrep.2020.100780 Text en © 2020 The Authors http://creativecommons.org/licenses/by/4.0/ This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Research Article
Asano, Teizo
Kaneko, Mika K.
Kato, Yukinari
RIEDL tag: A novel pentapeptide tagging system for transmembrane protein purification
title RIEDL tag: A novel pentapeptide tagging system for transmembrane protein purification
title_full RIEDL tag: A novel pentapeptide tagging system for transmembrane protein purification
title_fullStr RIEDL tag: A novel pentapeptide tagging system for transmembrane protein purification
title_full_unstemmed RIEDL tag: A novel pentapeptide tagging system for transmembrane protein purification
title_short RIEDL tag: A novel pentapeptide tagging system for transmembrane protein purification
title_sort riedl tag: a novel pentapeptide tagging system for transmembrane protein purification
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7369347/
https://www.ncbi.nlm.nih.gov/pubmed/32715101
http://dx.doi.org/10.1016/j.bbrep.2020.100780
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