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Development in Detection Methods for the Expression of Surface-Displayed Proteins
Directed evolution is a widely-used engineering strategy for improving the stabilities or biochemical functions of proteins by repeated rounds of mutation and selection. A protein of interest is selected as the template and expressed on a molecular display platform such as a bacteriophage for engine...
Autores principales: | , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
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Frontiers Media S.A.
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9085562/ https://www.ncbi.nlm.nih.gov/pubmed/35558116 http://dx.doi.org/10.3389/fmicb.2022.899578 |
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author | Ma, Chenglong Jiang, Chunyang Zhao, Dongping Li, Shuhao Li, Ronggui Li, Lei |
author_facet | Ma, Chenglong Jiang, Chunyang Zhao, Dongping Li, Shuhao Li, Ronggui Li, Lei |
author_sort | Ma, Chenglong |
collection | PubMed |
description | Directed evolution is a widely-used engineering strategy for improving the stabilities or biochemical functions of proteins by repeated rounds of mutation and selection. A protein of interest is selected as the template and expressed on a molecular display platform such as a bacteriophage for engineering. Initially, the surface-displayed protein template needs to be checked against the desired target via ELISA to examine whether the functions of the displayed template remain intact. The ELISA signal is subject to the protein-target binding affinity. A low-affinity results in a weak ELISA signal which makes it difficult to determine whether the weak signal is because of low affinity or because of poor expression of the protein. Using a methyllysine-binding chromodomain protein Cbx1 that weakly binds to the histone H3K9me3 peptide, we developed and compared three different approaches to increase the signal-to-background ratio of ELISA measurements. We observed that the specific peptide-binding signal was enhanced by increasing the Cbx1 phage concentration on the ELISA plate. The introduction of previously known gain-of-function mutations to the Cbx1 protein significantly increased the ELISA signals. Moreover, we demonstrated that the H3K9me3-specific binding signal was enhanced by fusing Cbx1 with a high-affinity phosphotyrosine-binding protein and by coating the ELISA plate with a mixture of H3K9me3 and phosphotyrosine peptides. This approach also worked with binding to a lower affinity momomethyllysine peptide H3K9me1. These approaches may help improve ELISA experiments when dealing with low-affinity ligand-protein interactions. |
format | Online Article Text |
id | pubmed-9085562 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | Frontiers Media S.A. |
record_format | MEDLINE/PubMed |
spelling | pubmed-90855622022-05-11 Development in Detection Methods for the Expression of Surface-Displayed Proteins Ma, Chenglong Jiang, Chunyang Zhao, Dongping Li, Shuhao Li, Ronggui Li, Lei Front Microbiol Microbiology Directed evolution is a widely-used engineering strategy for improving the stabilities or biochemical functions of proteins by repeated rounds of mutation and selection. A protein of interest is selected as the template and expressed on a molecular display platform such as a bacteriophage for engineering. Initially, the surface-displayed protein template needs to be checked against the desired target via ELISA to examine whether the functions of the displayed template remain intact. The ELISA signal is subject to the protein-target binding affinity. A low-affinity results in a weak ELISA signal which makes it difficult to determine whether the weak signal is because of low affinity or because of poor expression of the protein. Using a methyllysine-binding chromodomain protein Cbx1 that weakly binds to the histone H3K9me3 peptide, we developed and compared three different approaches to increase the signal-to-background ratio of ELISA measurements. We observed that the specific peptide-binding signal was enhanced by increasing the Cbx1 phage concentration on the ELISA plate. The introduction of previously known gain-of-function mutations to the Cbx1 protein significantly increased the ELISA signals. Moreover, we demonstrated that the H3K9me3-specific binding signal was enhanced by fusing Cbx1 with a high-affinity phosphotyrosine-binding protein and by coating the ELISA plate with a mixture of H3K9me3 and phosphotyrosine peptides. This approach also worked with binding to a lower affinity momomethyllysine peptide H3K9me1. These approaches may help improve ELISA experiments when dealing with low-affinity ligand-protein interactions. Frontiers Media S.A. 2022-04-25 /pmc/articles/PMC9085562/ /pubmed/35558116 http://dx.doi.org/10.3389/fmicb.2022.899578 Text en Copyright © 2022 Ma, Jiang, Zhao, Li, Li and Li. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. |
spellingShingle | Microbiology Ma, Chenglong Jiang, Chunyang Zhao, Dongping Li, Shuhao Li, Ronggui Li, Lei Development in Detection Methods for the Expression of Surface-Displayed Proteins |
title | Development in Detection Methods for the Expression of Surface-Displayed Proteins |
title_full | Development in Detection Methods for the Expression of Surface-Displayed Proteins |
title_fullStr | Development in Detection Methods for the Expression of Surface-Displayed Proteins |
title_full_unstemmed | Development in Detection Methods for the Expression of Surface-Displayed Proteins |
title_short | Development in Detection Methods for the Expression of Surface-Displayed Proteins |
title_sort | development in detection methods for the expression of surface-displayed proteins |
topic | Microbiology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9085562/ https://www.ncbi.nlm.nih.gov/pubmed/35558116 http://dx.doi.org/10.3389/fmicb.2022.899578 |
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