Column-free purification of an artificial protein nanocage, TIP60

Protein nanocages, which have inner cavities and surface pores, are attractive materials for various applications, such as in catalysts and medicine. Recently, we produced an artificial protein nanocage, TIP60, and demonstrated its potential as a stimuli-responsive nanocarrier. In the present study, we report a simple purification method for TIP60 that can replace time-consuming and costly affinity chromatography purification. TIP60, which has an anionic surface charge, aggregated at mildly acidic pH and redissolved at neutral pH, maintaining its cage structure. This pH-responsive reversible precipitation allowed us to purify TIP60 from soluble fractions of the E. coli cell lysate by controlling the pH. Compared with conventional Ni-NTA column purification, the pH-responsive precipitation method provided purified TIP60 with similar purity (∼80%) and higher yield. This precipitation purification method should facilitate the large-scale investigation and practical use of TIP60 nanocages.