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Transglutaminase 2 crosslinks the glutathione S-transferase tag, impeding protein–protein interactions of the fused protein
Glutathione S-transferase (GST) from Schistosoma japonicum has been widely used as a tag for affinity purification and pulldown of fusion proteins to detect protein–protein interactions. However, the reliability of this technique is undermined by the formation of GST-fused protein aggregates after i...
Autores principales: | , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
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Nature Publishing Group UK
2021
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8080825/ https://www.ncbi.nlm.nih.gov/pubmed/33441971 http://dx.doi.org/10.1038/s12276-020-00549-9 |
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author | Kim, Hyo-Jun Lee, Jin-Haeng Lee, Ki Baek Shin, Ji-Woong Kwon, Mee-ae Lee, Soojin Jeong, Eui Man Cho, Sung-Yup Kim, In-Gyu |
author_facet | Kim, Hyo-Jun Lee, Jin-Haeng Lee, Ki Baek Shin, Ji-Woong Kwon, Mee-ae Lee, Soojin Jeong, Eui Man Cho, Sung-Yup Kim, In-Gyu |
author_sort | Kim, Hyo-Jun |
collection | PubMed |
description | Glutathione S-transferase (GST) from Schistosoma japonicum has been widely used as a tag for affinity purification and pulldown of fusion proteins to detect protein–protein interactions. However, the reliability of this technique is undermined by the formation of GST-fused protein aggregates after incubation with cell lysates. It remains unknown why this aggregation occurs. Here, we demonstrate that the GST tag is a substrate of transglutaminase 2 (TG2), which is a calcium-dependent enzyme that polyaminates or crosslinks substrate proteins. Mutation analysis identified four glutamine residues in the GST tag as polyamination sites. TG2-mediated modification of the GST tag caused aggregate formation but did not affect its glutathione binding affinity. When incubated with cell lysates, GST tag aggregation was dependent on cellular TG2 expression levels. A GST mutant in which four glutamine residues were replaced with asparagine (GST(4QN)) exhibited a glutathione binding affinity similar to that of wild-type GST and could be purified by glutathione affinity chromatography. Moreover, the use of GST(4QN) as a tag reduced fused p53 aggregation and enhanced the induction of p21 transcription and apoptosis in cells treated with 5-fluorouracil (5-FU). These results indicated that TG2 interferes with the protein–protein interactions of GST-fused proteins by crosslinking the GST tag; therefore, a GST(4QN) tag could improve the reproducibility and reliability of GST pulldown experiments. |
format | Online Article Text |
id | pubmed-8080825 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | Nature Publishing Group UK |
record_format | MEDLINE/PubMed |
spelling | pubmed-80808252021-04-29 Transglutaminase 2 crosslinks the glutathione S-transferase tag, impeding protein–protein interactions of the fused protein Kim, Hyo-Jun Lee, Jin-Haeng Lee, Ki Baek Shin, Ji-Woong Kwon, Mee-ae Lee, Soojin Jeong, Eui Man Cho, Sung-Yup Kim, In-Gyu Exp Mol Med Article Glutathione S-transferase (GST) from Schistosoma japonicum has been widely used as a tag for affinity purification and pulldown of fusion proteins to detect protein–protein interactions. However, the reliability of this technique is undermined by the formation of GST-fused protein aggregates after incubation with cell lysates. It remains unknown why this aggregation occurs. Here, we demonstrate that the GST tag is a substrate of transglutaminase 2 (TG2), which is a calcium-dependent enzyme that polyaminates or crosslinks substrate proteins. Mutation analysis identified four glutamine residues in the GST tag as polyamination sites. TG2-mediated modification of the GST tag caused aggregate formation but did not affect its glutathione binding affinity. When incubated with cell lysates, GST tag aggregation was dependent on cellular TG2 expression levels. A GST mutant in which four glutamine residues were replaced with asparagine (GST(4QN)) exhibited a glutathione binding affinity similar to that of wild-type GST and could be purified by glutathione affinity chromatography. Moreover, the use of GST(4QN) as a tag reduced fused p53 aggregation and enhanced the induction of p21 transcription and apoptosis in cells treated with 5-fluorouracil (5-FU). These results indicated that TG2 interferes with the protein–protein interactions of GST-fused proteins by crosslinking the GST tag; therefore, a GST(4QN) tag could improve the reproducibility and reliability of GST pulldown experiments. Nature Publishing Group UK 2021-01-13 /pmc/articles/PMC8080825/ /pubmed/33441971 http://dx.doi.org/10.1038/s12276-020-00549-9 Text en © The Author(s) 2021 https://creativecommons.org/licenses/by/4.0/Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) . |
spellingShingle | Article Kim, Hyo-Jun Lee, Jin-Haeng Lee, Ki Baek Shin, Ji-Woong Kwon, Mee-ae Lee, Soojin Jeong, Eui Man Cho, Sung-Yup Kim, In-Gyu Transglutaminase 2 crosslinks the glutathione S-transferase tag, impeding protein–protein interactions of the fused protein |
title | Transglutaminase 2 crosslinks the glutathione S-transferase tag, impeding protein–protein interactions of the fused protein |
title_full | Transglutaminase 2 crosslinks the glutathione S-transferase tag, impeding protein–protein interactions of the fused protein |
title_fullStr | Transglutaminase 2 crosslinks the glutathione S-transferase tag, impeding protein–protein interactions of the fused protein |
title_full_unstemmed | Transglutaminase 2 crosslinks the glutathione S-transferase tag, impeding protein–protein interactions of the fused protein |
title_short | Transglutaminase 2 crosslinks the glutathione S-transferase tag, impeding protein–protein interactions of the fused protein |
title_sort | transglutaminase 2 crosslinks the glutathione s-transferase tag, impeding protein–protein interactions of the fused protein |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8080825/ https://www.ncbi.nlm.nih.gov/pubmed/33441971 http://dx.doi.org/10.1038/s12276-020-00549-9 |
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