title: Mutational analysis of protein solubility enhancement using short peptide tags.
abstract: 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.
authors.affiliation: Department of Biotechnology and Life Sciences, Graduate School of Engineering, Tokyo University of Agriculture and Technology, 2-24-16 Nakamachi, Koganei-shi, Tokyo 184-8588, Japan.
authors.forename: Atsushi
authors.initials: A
authors.lastname: Kato
authors.forename: Kosuke
authors.initials: K
authors.lastname: Maki
authors.forename: Teppei
authors.initials: T
authors.lastname: Ebina
authors.forename: Kunihiro
authors.initials: K
authors.lastname: Kuwajima
authors.forename: Kunitsugu
authors.initials: K
authors.lastname: Soda
authors.forename: Yutaka
authors.initials: Y
authors.lastname: Kuroda
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D007611:Aprotinin / Q000737:chemistry* / Q000235:genetics; D001120:Arginine / Q000737:chemistry; D002417:Cattle; D008239:Lysine / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000737:chemistry*; D012995:Solubility
chemical_list: D010455:Peptides; C015462:polyarginine; D007611:Aprotinin; D001120:Arginine; D008239:Lysine