Engineered oligomeric tags confer cation responsiveness and reversible precipitation ability to DsRed, facilitating
Hongxu Sun1, Chao Gao1, Ruping Lu1
1Department of Biological Science and Engineering, School of Chemistry and Biological Engineering, University of Science and Technology Beijing, Beijing, 100083, China.
Abstract:
Fusion tags and intrinsic surface residues on proteins interact intricately with ions in solution. These interactions can be harnessed to enable protein purification, while uncontrolled interactions may lead to undesired aggregation or precipitation. In this study, we systematically investigated the cation-responsive behaviors of oligopeptide tags, composed of single or mixed amino acid types, by fusing them to DsRed. Our results showed that basic and aromatic amino acids induced strong precipitation in the presence of Mg2+ and Na+, amide-containing amino acids exhibited moderate responses, while acidic and aliphatic residues showed no response or even negative response. Hybrid tags combining strongly responsive residues, such as histidine (H), with moderately responsive residues, such as glutamine (Q), markedly shifted the precipitation response curves toward lower salt concentrations. Based on this observation, we developed the (HQ)3 tag, which enables efficient cation affinity purification under low-salt conditions. This work not only characterizes the ion-responsiveness of various amino acid types, but also provides a new strategy for designing precipitation-based purification tags and for achieving controllable protein precipitation and solubilization through surface residue engineering.
