A phase separation process induced by pH change for purification of His-tagged protein at low salt concentration

Abstract

Protein purification is critical for protein research and enzyme manufacture, and efficient purification methods with low cost have a high priority. In this study, the difference in solubility between His-tagged protein and nontagged protein led to a liquid-solid phase separation at neutral pH and low salt concentration and purified His-tagged protein to some extent. The investigation of impurity in the precipitate indicated that solvation behavior varied from several oligo-amino acid tagged proteins. Among them, proteins with oligo-Lys, oligo-Trp and oligo-Arg obviously precipitated at low salt concentration and weak acidic pH, which is similar to that for the precipitation of His-tagged protein. Further study suggested that proteins with oligo-Lys, oligo-Trp and oligo-Arg remained a precipitate state while His-tagged protein dissolved at a slightly higher pH value. Based on these finding, a method was developed for the purification of His-tagged protein via phase separation induced by pH (PSIP) at low salt concentration. Though the yield and purity of protein purified by PSIP were slightly inferior to that by immobilized metal ion affinity chromatography (IMAC), PSIP was a high-throughput and low-cost method for purification of His-tagged proteins. Beyond the purification process, the various solvation behaviors of proteins with different oligo-amino acid also provide a reference for solvation research of protein.