Codon Harmonization of a Kir3.1-KirBac1.3 Chimera for Structural Study Optimization

Evan Van Aalst,Anil K Mehta,Isaac Eason,Benjamin Wylie,Maryam Yekefallah

doi:10.3390/biom10030430

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.

Highlights

Solid-state nuclear magnetic resonance (SSNMR) is a key structural technique to study membrane proteins within their native or native-like bilayer environment
While many SSNMR studies quantify the structure and dynamics of membrane proteins within bilayers, the advancement of this technology is often limited by sample availability: the quantity of protein produced per gram of 15N, 13C, and/or 2H enriched material is a major limiting factor for NMR studies
We show how applications of codon harmonization can positively influence the yield of membrane proteins for NMR and functional studies

Summary

Introduction

Solid-state nuclear magnetic resonance (SSNMR) is a key structural technique to study membrane proteins within their native or native-like bilayer environment. Membrane protein function strongly depends upon the composition and phase of the surrounding lipids, further necessitating structural studies within the bilayer environment at physiological temperature. While many SSNMR studies quantify the structure and dynamics of membrane proteins within bilayers, the advancement of this technology is often limited by sample availability: the quantity of protein produced per gram of 15N, 13C, and/or 2H enriched material is a major limiting factor for NMR studies. We present an extensive study of codon harmonization in expression of a membrane protein from minimal media, and show we produce large quantities of functional, folded protein. The wider use of codon harmonization in the NMR community could be another powerful tool to aid in the ever-necessary quest to increase recombinant protein expression for structural studies

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