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.

Structure–Activity Relationship and Molecular Docking of a Kunitz-Like Trypsin Inhibitor, Kunitzin-AH, from the Skin Secretion of Amolops hainanensis

“…The system was subjected to a total of 100 ns of molecular equilibration dynamics, with an integration time of 2 fs. A Langevin dynamics approach was conducted to retain the temperature of the system at 298 K [ 35 , 36 , 37 ]. The structures of AH-837 and AH-884 were modified based on that of AH-798.…”

Cholesterol Is a Dose-Dependent Positive Allosteric Modulator of CCR3 Ligand Affinity and G Protein Coupling

“…Furthermore, we employed codon harmonization, a method to optimize heterologous plasmid DNA sequences ( Angov et al, 2008 ). We previously showed our codon harmonization strategy, dubbed DNA codon usage for measured base optimization, or DUMB optimization (DO), which dramatically increased both the yield and activity of an exogenously expressed chimeric membrane protein ( van Aalst et al, 2020 ). Results of %MinMax analysis of proteins in this work show the codon usage of the fully optimized and codon-harmonized constructs as compared to native usage ( Figure 3 ).…”

“…The native human CCR3 DNA sequence was obtained from the European Nucleotide Archive (ENA, Sequence: U51241) and codon-harmonized according to DUMB optimization (DO) for expression in E. coli , as previously described ( van Aalst et al . , 2020 ).…”

“…Human and E. coli codon usage frequencies from the Graphical Codon Usage Analyzer ( http://gcua.schoedl.de/ ) were used in designing the DO gene sequence ( Fuhrmann et al, 2004 ). For more information on the codon harmonization process, see reference van Aalst et al, 2020 . %MinMax analysis ( Rodriguez et al .…”

“…In pursuit of this goal, we implemented a codon harmonization scheme we previously reported to positively influence heterologous membrane protein yield ( van Aalst et al, 2020 ). This technique is called DNA codon usage for measured base optimization, or DUMB optimization (DO).…”