Mutational analysis of protein solubility enhancement using short peptide tags

Protein aggregation is a common phenomenon. The preparation of highly concentrated protein samples, typically required for biophysical measurements, often involves a time consuming and tedious testing of solvent conditions for improving protein solubility. Here, in a systematic analysis, we have determined the increase in solubility upon the addition of SEP‐tags (solubility enhancement peptide tags) containing, one, three, and five lysines or arginines (or six arginines) to either the N or C terminus of our low solubility model protein, bovine pancreatic trypsin inhibitor variant, BPTI‐22 (a BPTI variant containing 22 alanines). As anticipated, the BPTI‐22 solubility increased in direct relation to the number of charged residues contained in the SEP‐tag, and without altering either the activity or the structure of the protein. The largest solubility increases were of 4.2‐, 4.8‐, and 6.2‐folds produced by the addition, at the C terminus, of five lysine (BPTI‐22‐C5K), five and six arginine residues (BPTI‐22‐C5R and BPTI‐22‐C6R), respectively. The increased solubility of the tagged BPTI‐22 yielded higher quality NMR spectra (hetero single quantum correlation HSQC spectra; with respect of the signal‐to‐noise and line shapes) in a much shorter time than for the untagged BPTI‐22. Furthermore, tagged samples remained soluble for over ten days, as observed by their HSQC spectra. We believe that lysine‐ and arginine‐based SEP‐tags may provide an effective and versatile method for enhancing protein solubility. © 2006 Wiley Periodicals, Inc. Biopolymers 85: 12–18, 2007. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com