Codon harmonization of a kir3.1-kirbac1.3 chimera for structural study optimization

Abstract

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.

Description

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. cc-by

Keywords

Codon bias, Codon harmonization, K+channels, Nuclear magnetic resonance (NMR), Protein expression, Protein folding

Citation

Van, Aalst, E., Yekefallah, M., Mehta, A.K., Eason, I., & Wylie, B.. 2020. Codon harmonization of a kir3.1-kirbac1.3 chimera for structural study optimization. Biomolecules, 10(3). https://doi.org/10.3390/biom10030430