Abstract
Synonymous codons occur with different frequencies in different organisms, a phenomenon termed codon usage bias. Codon optimization, a common term for a variety of approaches used widely by the biopharmaceutical industry, involves synonymous substitutions to increase protein expression. It had long been presumed that synonymous variants, which, by definition, do not alter the primary amino acid sequence, have no effect on protein structure and function. However, a critical mass of reports suggests that synonymous codon variations may impact protein conformation. To investigate the impact of synonymous codons usage on protein expression and function, we designed an optimized coagulation factor IX (FIX) variant and used multiple methods to compare its properties to the wild-type FIX upon expression in HEK293T cells. We found that the two variants differ in their conformation, even when controlling for the difference in expression levels. Using ribosome profiling, we identified robust changes in the translational kinetics of the two variants and were able to identify a region in the gene that may have a role in altering the conformation of the protein. Our data have direct implications for codon optimization strategies, for production of recombinant proteins and gene therapies.
Highlights
The genetic code is redundant, with most amino acids being encoded by more than one codon, some by as many as six
We used human blood coagulation factor IX (FIX, when referring to the protein and F9 when referring to the gene), as a model to study the effects of codon optimization on the kinetics of protein translation and protein conformation
Codon optimization leads to the omission of rare codons and enrichment of common ones
Summary
The genetic code is redundant, with most amino acids being encoded by more than one (synonymous) codon, some by as many as six. Ribosome profiling[29] allows the study of translation kinetics at single-codon resolution, opening a window of opportunity for identifying regions on the gene where synonymous substitutions are most likely to alter protein conformation. It should be noted, that early ribosome profiling experiments did not reveal a clear correlation between ribosomal stalling and rare codons[30]. Following recent improvements in the ribosome profiling method[35], a deep-learning based approach was successful in predicting ribosome stalling and correlating it with codon usage, as well as with tRNA adaptation, codon co-occurrence, proline codons, mRNAN6-methyladenosine modification, RNA-binding proteins and protein secondary structure, further pointing to the complexity of the association[36]. The improved understanding of the effect of codon optimization on protein conformation that we have gained from this study may contribute to the development of safer and more efficient FIX therapeutics
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