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CP5 system, for simple and highly efficient protein purification with a C-terminal designed mini tag
There are many strategies to purify recombinant proteins of interest, and affinity purification utilizing monoclonal antibody that targets a linear epitope sequence is one of the essential techniques used in current biochemistry and structural biology. Here we introduce a new protein purification sy...
Autores principales: | , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2017
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5444806/ https://www.ncbi.nlm.nih.gov/pubmed/28542437 http://dx.doi.org/10.1371/journal.pone.0178246 |
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author | Takeda, Hiroyuki Zhou, Wei Kido, Kohki Suno, Ryoji Iwasaki, Takahiro Kobayashi, Takuya Sawasaki, Tatsuya |
author_facet | Takeda, Hiroyuki Zhou, Wei Kido, Kohki Suno, Ryoji Iwasaki, Takahiro Kobayashi, Takuya Sawasaki, Tatsuya |
author_sort | Takeda, Hiroyuki |
collection | PubMed |
description | There are many strategies to purify recombinant proteins of interest, and affinity purification utilizing monoclonal antibody that targets a linear epitope sequence is one of the essential techniques used in current biochemistry and structural biology. Here we introduce a new protein purification system using a very short CP5 tag. First, we selected anti-dopamine receptor D1 (DRD1) rabbit monoclonal antibody clone Ra62 (Ra62 antibody) as capture antibody, and identified its minimal epitope sequence as a 5-amino-acid sequence at C-terminal of DRD1 (GQHPT-COOH, D1CE sequence). We found that single amino acid substitution in D1CE sequence (GQHVT-COOH) increased dissociation rate up to 10-fold, and named the designed epitope sequence CP5 tag. Using Ra62 antibody and 2 peptides with different affinity, we developed a new affinity protein purification method, CP5 system. Ra62 antibody quickly captures CP5-tagged target protein, and captured CP5-tagged protein was eluted by competing with higher affinity D1CE peptide. By taking the difference of the affinity between D1CE and CP5, sharp elution under mild condition was achieved. Using CP5 system, we successfully purified deubiquitinase CYLD and E3 ubiquitin ligase MARCH3, and detected their catalytic activity. As to G protein-coupled receptors (GPCRs), 9 out of 12 cell-free synthesized ones were purified, demonstrating its purification capability of integral membrane proteins. CP5 tagged CHRM2 expressed by baculovirus-insect cell was also successfully purified by CP5 system. CP5 system offers several distinct advantages in addition to its specificity and elution performance. CP5 tag is easy to construct and handle because of its short length, which has less effect on protein characters. Mild elution of CP5 system is particulaly suitable for preparing delicate proteins such as enzymes and membrane proteins. Our data demonstrate that CP5 system provides a new promising option in protein sample preparation with high yield, purity and activity for downstream applications in functional and structural analysis. |
format | Online Article Text |
id | pubmed-5444806 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2017 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-54448062017-06-12 CP5 system, for simple and highly efficient protein purification with a C-terminal designed mini tag Takeda, Hiroyuki Zhou, Wei Kido, Kohki Suno, Ryoji Iwasaki, Takahiro Kobayashi, Takuya Sawasaki, Tatsuya PLoS One Research Article There are many strategies to purify recombinant proteins of interest, and affinity purification utilizing monoclonal antibody that targets a linear epitope sequence is one of the essential techniques used in current biochemistry and structural biology. Here we introduce a new protein purification system using a very short CP5 tag. First, we selected anti-dopamine receptor D1 (DRD1) rabbit monoclonal antibody clone Ra62 (Ra62 antibody) as capture antibody, and identified its minimal epitope sequence as a 5-amino-acid sequence at C-terminal of DRD1 (GQHPT-COOH, D1CE sequence). We found that single amino acid substitution in D1CE sequence (GQHVT-COOH) increased dissociation rate up to 10-fold, and named the designed epitope sequence CP5 tag. Using Ra62 antibody and 2 peptides with different affinity, we developed a new affinity protein purification method, CP5 system. Ra62 antibody quickly captures CP5-tagged target protein, and captured CP5-tagged protein was eluted by competing with higher affinity D1CE peptide. By taking the difference of the affinity between D1CE and CP5, sharp elution under mild condition was achieved. Using CP5 system, we successfully purified deubiquitinase CYLD and E3 ubiquitin ligase MARCH3, and detected their catalytic activity. As to G protein-coupled receptors (GPCRs), 9 out of 12 cell-free synthesized ones were purified, demonstrating its purification capability of integral membrane proteins. CP5 tagged CHRM2 expressed by baculovirus-insect cell was also successfully purified by CP5 system. CP5 system offers several distinct advantages in addition to its specificity and elution performance. CP5 tag is easy to construct and handle because of its short length, which has less effect on protein characters. Mild elution of CP5 system is particulaly suitable for preparing delicate proteins such as enzymes and membrane proteins. Our data demonstrate that CP5 system provides a new promising option in protein sample preparation with high yield, purity and activity for downstream applications in functional and structural analysis. Public Library of Science 2017-05-25 /pmc/articles/PMC5444806/ /pubmed/28542437 http://dx.doi.org/10.1371/journal.pone.0178246 Text en © 2017 Takeda et al http://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. |
spellingShingle | Research Article Takeda, Hiroyuki Zhou, Wei Kido, Kohki Suno, Ryoji Iwasaki, Takahiro Kobayashi, Takuya Sawasaki, Tatsuya CP5 system, for simple and highly efficient protein purification with a C-terminal designed mini tag |
title | CP5 system, for simple and highly efficient protein purification with a C-terminal designed mini tag |
title_full | CP5 system, for simple and highly efficient protein purification with a C-terminal designed mini tag |
title_fullStr | CP5 system, for simple and highly efficient protein purification with a C-terminal designed mini tag |
title_full_unstemmed | CP5 system, for simple and highly efficient protein purification with a C-terminal designed mini tag |
title_short | CP5 system, for simple and highly efficient protein purification with a C-terminal designed mini tag |
title_sort | cp5 system, for simple and highly efficient protein purification with a c-terminal designed mini tag |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5444806/ https://www.ncbi.nlm.nih.gov/pubmed/28542437 http://dx.doi.org/10.1371/journal.pone.0178246 |
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