(707d) Enhancing Protein Solubility By Rationally Programming Co-Translational Folding Using Synonymous Codons

Biological production of active proteins requires soluble expression. However, few techniques exist to improve protein solubility, largely because solubility optimization is believed to be protein-dependent, difficult to predict computationally, and more challenging for more complex proteins. 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 work, we have explored the introduction of synthetic transient pauses between structural domains in a heterologous model protein based on computationally designed patterns of synonymous codons that affect the affinity between the mRNA and the anti-Shineâ??Dalgarno (aSD) sequence on the ribosome. Although synonymous codon sequences do not affect the amino acid identity of a protein, they have demonstrated profound effects on protein function. Specifically, we have demonstrated that optimizing translation attenuation at domain boundaries can predictably affect solubility patterns in bacteria; in these studies attenuation is based on computationally designed increased affinity (i.e. binding energies) between the ribosomal anti-Shine-Dalgarno sequence and the messenger RNA. Exploration of the affinity space showed that modifying less than 1% of the nucleotides in short interdomain amino acid linkers can vary soluble protein yields up to â?¼7-fold without altering the primary sequence of the protein. Given our findings that interdomain ribosomal affinity can affect protein solubility in a predictable manner, we have also explored computational design of protein solubility by exploiting ribosomal affinities in combination with other parameters that include mRNA structure, and tRNA abundance, also known to influence nascent protein fate. Based on a proteinâ??s secondary structure, we hypothesized key regions where transient pausing can provide the greatest enhancement in solubility. Highly structured, high affinity, and/or low tRNA abundance synonymous mutations were implemented in these regions. Conversely, relatively unstructured, low affinity, and high abundance codons were used in regions where enhanced translation would not lead to detrimental interactions between nascent chain fragments. In this talk our results on the rational design of key factors that modulate translation timing as a novel protein engineering strategy will be discussed.