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Codon Harmonization of a Kir3.1-KirBac1.3 Chimera for Structural Study Optimization
The expression of functional, folded, and isotopically enriched membrane proteins is an enduring bottleneck for nuclear magnetic resonance (NMR) studies. Indeed, historically, protein yield optimization has been insufficient to allow NMR analysis of many complex Eukaryotic membrane proteins. However...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2020
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7175280/ https://www.ncbi.nlm.nih.gov/pubmed/32164257 http://dx.doi.org/10.3390/biom10030430 |
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author | Van Aalst, Evan Yekefallah, Maryam Mehta, Anil K. Eason, Isaac Wylie, Benjamin |
author_facet | Van Aalst, Evan Yekefallah, Maryam Mehta, Anil K. Eason, Isaac Wylie, Benjamin |
author_sort | Van Aalst, Evan |
collection | PubMed |
description | The expression of functional, folded, and isotopically enriched membrane proteins is an enduring bottleneck for nuclear magnetic resonance (NMR) studies. Indeed, historically, protein yield optimization has been insufficient to allow NMR analysis of many complex Eukaryotic membrane proteins. However, recent work has found that manipulation of plasmid codons improves the odds of successful NMR-friendly protein production. In the last decade, numerous studies showed that matching codon usage patterns in recombinant gene sequences to those in the native sequence is positively correlated with increased protein yield. This phenomenon, dubbed codon harmonization, may be a powerful tool in optimizing recombinant expression of difficult-to-produce membrane proteins for structural studies. Here, we apply this technique to an inward rectifier K(+) Channel (Kir) 3.1-KirBac1.3 chimera. Kir3.1 falls within the G protein-coupled inward rectifier K(+) (GIRK) channel family, thus NMR studies may inform on the nuances of GIRK gating action in the presence and absence of its G Protein, lipid, and small molecule ligands. In our hands, harmonized plasmids increase protein yield nearly two-fold compared to the traditional ‘fully codon optimized’ construct. We then employ a fluorescence-based functional assay and solid-state NMR correlation spectroscopy to show the final protein product is folded and functional. |
format | Online Article Text |
id | pubmed-7175280 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2020 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-71752802020-04-28 Codon Harmonization of a Kir3.1-KirBac1.3 Chimera for Structural Study Optimization Van Aalst, Evan Yekefallah, Maryam Mehta, Anil K. Eason, Isaac Wylie, Benjamin Biomolecules Article The expression of functional, folded, and isotopically enriched membrane proteins is an enduring bottleneck for nuclear magnetic resonance (NMR) studies. Indeed, historically, protein yield optimization has been insufficient to allow NMR analysis of many complex Eukaryotic membrane proteins. However, recent work has found that manipulation of plasmid codons improves the odds of successful NMR-friendly protein production. In the last decade, numerous studies showed that matching codon usage patterns in recombinant gene sequences to those in the native sequence is positively correlated with increased protein yield. This phenomenon, dubbed codon harmonization, may be a powerful tool in optimizing recombinant expression of difficult-to-produce membrane proteins for structural studies. Here, we apply this technique to an inward rectifier K(+) Channel (Kir) 3.1-KirBac1.3 chimera. Kir3.1 falls within the G protein-coupled inward rectifier K(+) (GIRK) channel family, thus NMR studies may inform on the nuances of GIRK gating action in the presence and absence of its G Protein, lipid, and small molecule ligands. In our hands, harmonized plasmids increase protein yield nearly two-fold compared to the traditional ‘fully codon optimized’ construct. We then employ a fluorescence-based functional assay and solid-state NMR correlation spectroscopy to show the final protein product is folded and functional. MDPI 2020-03-10 /pmc/articles/PMC7175280/ /pubmed/32164257 http://dx.doi.org/10.3390/biom10030430 Text en © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Van Aalst, Evan Yekefallah, Maryam Mehta, Anil K. Eason, Isaac Wylie, Benjamin Codon Harmonization of a Kir3.1-KirBac1.3 Chimera for Structural Study Optimization |
title | Codon Harmonization of a Kir3.1-KirBac1.3 Chimera for Structural Study Optimization |
title_full | Codon Harmonization of a Kir3.1-KirBac1.3 Chimera for Structural Study Optimization |
title_fullStr | Codon Harmonization of a Kir3.1-KirBac1.3 Chimera for Structural Study Optimization |
title_full_unstemmed | Codon Harmonization of a Kir3.1-KirBac1.3 Chimera for Structural Study Optimization |
title_short | Codon Harmonization of a Kir3.1-KirBac1.3 Chimera for Structural Study Optimization |
title_sort | codon harmonization of a kir3.1-kirbac1.3 chimera for structural study optimization |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7175280/ https://www.ncbi.nlm.nih.gov/pubmed/32164257 http://dx.doi.org/10.3390/biom10030430 |
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