ClinicalTrials.gov: http://clinicaltrials.gov/show/NCT01359878. Published protocol: http://www.trialsjournal.com/content/pdf/1745-6215-13-110.pdf.

While the field of computational protein design has witnessed amazing progression in recent years, folding properties still constitute a significant barrier towards designing new and larger proteins. In order to assess and improve folding properties of designed proteins, we have developed a genetics-based folding assay and selection system based on the essential enzyme, orotate phosphoribosyl transferase from Escherichia coli. This system allows for both screening of candidate designs with good folding properties and genetic selection of improved designs. Thus, we identified single amino acid substitutions in two failed designs that rescued poorly folding and unstable proteins. Furthermore, when these substitutions were transferred into a well-structured design featuring a complex folding profile, the resulting protein exhibited native-like cooperative folding with significantly improved stability. In protein design, a single amino acid can make the difference between folding and misfolding, and this approach provides a useful new platform to identify and improve candidate designs.

Alginates, serving as hydrocolloids in the food and pharma industries, form particles at pH < 4.5 with positively charged proteins, such as β-lactoglobulin (β-Lg). Alginates are linear anionic polysaccharides composed of 1,4-linked β-d-mannuronate (M) and α-l-guluronate (G) residues. The impact of M and G contents and pH is investigated to correlate with the formation and size of β-Lg alginate complexes under relevant ionic strength. It is concluded, using three alginates of M/G ratios 0.6, 1.1, and 1.8 and similar molecular mass, that β-Lg binding capacity is higher at pH 4.0 than at pH 2.65 and for high M content. By contrast, the largest particles are obtained at pH 2.65 and with high G content. At pH 4.0 and 2.65, the stoichiometry was 28–48 and 3–10 β-Lg molecules bound per alginate, respectively, increasing with higher M content. The findings will contribute to the design of formation of the desired alginate–protein particles in the acidic pH range.

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