Analysis of amino acid contributions to protein solubility using short peptide tags fused to a simplified BPTI variant.

Protein solubility is usually characterized in terms of a hydrophobicity scale, which refers to the free energy of transfer of a molecule from an aqueous to a nonpolar solution and is not a "solubility propensity scale" per se. Using a "host-guest" approach, we measured the effects of short poly-amino-acid tags (guests) on the solubility of a host protein, a simplified bovine pancreatic trypsin inhibitor (BPTI), to which they were fused at the C-terminus. We analyzed 10 amino acid types, representing the full range of biophysical properties (acidic, basic, polar, and hydrophobic). As anticipated, positively charged residues significantly increased the solubility of the model protein, at both pH 4.7 and 7.7, whereas very hydrophobic poly-Ile markedly reduced the solubility of BPTI. Poly-Asp and poly-Glu barely affected BPTI solubility at pH 4.7, but induced an eight to ten-fold increase at pH 7.7, attributable to the ionization of their side chains. Although Pro is the most soluble amino acid, poly-Pro did not affect the protein's solubility. The effects of the other tags on BPTI solubility ranged from none to an eight-fold increase. To ensure that the measured solubility values were context independent and could provide a "solubility propensity scale", we confirmed that the tags remained independent of the structure, thermal stability, and biochemical activity of the host protein. These observations suggest that this approach is valuable for measuring the solubility propensities of amino acids, which could eventually allow the calculation of a polypeptide's relative solubility from its amino acid sequence.