Slowing Translation between Protein Domains by Increasing Affinity between mRNAs and the Ribosomal Anti-Shine–Dalgarno Sequence Improves Solubility

Abstract

Recent studies have demonstrated that effective protein production requires coordination of multiple cotranslational cellular processes, which are heavily affected by translation timing. Until recently, protein engineering has focused on codon optimization to maximize protein production rates, mostly considering the effect of tRNA abundance. However, as it relates to complex multidomain proteins, it has been hypothesized that strategic translational pauses between domains and between distinct individual structural motifs can prevent interactions between nascent chain fragments that generate kinetically trapped misfolded peptides and thereby enhance protein yields. In this study, we introduce synthetic transient pauses between structural domains in a heterologous model protein based on designed patterns of affinity between the mRNA and the anti-Shine-Dalgarno (aSD) sequence on the ribosome. We demonstrate that optimizing translation attenuation at domain boundaries can predictably affect solubility patterns in bacteria. Exploration of the affinity space showed that modifying less than 1% of the nucleotides (on a small 12 amino acid linker) can vary soluble protein yields up to ∼7-fold without altering the primary sequence of the protein. In the context of longer linkers, where a larger number of distinct structural motifs can fold outside the ribosome, optimal synonymous codon variations resulted in an additional 2.1-fold increase in solubility, relative to that of nonoptimized linkers of the same length. While rational construction of 54 linkers of various affinities showed a significant correlation between protein solubility and predicted affinity, only weaker correlations were observed between tRNA abundance and protein solubility. We also demonstrate that naturally occurring high-affinity clusters are present between structural domains of β-galactosidase, one of Escherichia coli's largest native proteins. Interdomain ribosomal affinity is an important factor that has not previously been explored in the context of protein engineering.

Quality over quantity: optimizing co-translational protein folding with non-‘optimal’ synonymous codons

“…Although their impact on translation rate in vivo now appears smaller than previously thought [25,71], internal SD sequences are disfavored in E. coli coding sequences [24], possibly due to their negative effect on translation elongation rate. Consistent with this, introducing SD sequences within coding sequences has been shown to negatively impact protein accumulation [72]. …”

Differential bicodon usage in lowly and highly abundant proteins

“…This is due to several constraints operating to optimize many important biological features such as expression level (Guimaraes, Rocha & Arkin, 2014), translational accuracy (Zaher & Green, 2009), protein solubility (Vasquez et al, 2016), folding accuracy (Sauna & Kimchi-Sarfaty, 2011; Spencer et al, 2012; Bali & Bebok, 2015), and protein stability, among others. In this sense, it has been shown that codon usage in E. coli is biased to reduce the cost of translational errors (Stoletzki & Eyre-Walker, 2007).…”

Depletion of Shine-Dalgarno Sequences Within Bacterial Coding Regions Is Expression Dependent

“…Owing to the compact nature of bacterial genomes, the translation initiation site of many genes within operons will occur within the 3′ terminus of the preceding coding sequence. Further, the presence of multiple translation initiation sites may serve a regulatory role for certain proteins, allowing for the production of distinct isoforms depending on the N-terminal sequence or controlling protein folding rates (Ozin et al 2001; Fluman et al 2014; Schrader et al 2014; Vasquez et al 2015). …”

“…SD sequence-mediated pauses have now been documented for several bacterial species and independent ribosomal profiling datasets (Li et al 2012; Liu et al 2013; Schrader et al 2014). Studies have built on these results by showing SD-associated pauses in vitro , negative effects of SD sequences on protein production in engineered sequences, enhanced solubility of recombinant proteins via rational insertion of SD sequences at protein domain boundaries, and enrichment of SD sequences following transmembrane domains of natural sequences (Agashe et al 2013; Chen et al 2014; Chevance et al 2014; Fluman et al 2014; Vasquez et al 2015). …”