Codon Harmonization of a Kir3.1-KirBac1.3 Chimera for Structural Study Optimization
| Autor: | Anil K. Mehta, Isaac R. Eason, Benjamin J. Wylie, Maryam Yekefallah, Evan van Aalst |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2020 |
| Předmět: |
Protein Folding
G protein nuclear magnetic resonance (NMR) Recombinant Fusion Proteins lcsh:QR1-502 Computational biology Biochemistry lcsh:Microbiology Article 03 medical and health sciences k+ channels codon bias Protein biosynthesis Escherichia coli Humans G protein-coupled inwardly-rectifying potassium channel Molecular Biology protein expression Nuclear Magnetic Resonance Biomolecular 030304 developmental biology 0303 health sciences codon harmonization Chemistry 030302 biochemistry & molecular biology Small molecule Membrane protein G Protein-Coupled Inwardly-Rectifying Potassium Channels Codon usage bias Protein folding K+ channels Two-dimensional nuclear magnetic resonance spectroscopy |
| Zdroj: | Biomolecules Volume 10 Issue 3 Biomolecules, Vol 10, Iss 3, p 430 (2020) |
| ISSN: | 2218-273X |
| Popis: | 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 &lsquo fully codon optimized&rsquo 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. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|
| Nepřihlášeným uživatelům se plný text nezobrazuje | K zobrazení výsledku je třeba se přihlásit. |