A Novel Recombinant Protein Purification Approach Using Biomolecular Condensates

The lipoate-protein ligase A (LplA) identified in Escherichia coli K-12 exhibits structural homomeric oligomerization and reversible lower critical solution temperature (LCST)-type phase separation in vitro. In this study, based on the ability of LplA to form condensates, it was utilized as a temperature-sensitive purification tag in the field of protein purification for the first time, and a novel and convenient one-step purification method was established. A universal vector was developed for the fusion expression of LplA and the target protein. The fusion protein forms condensates upon heating, separating from the solution, and redissolves in buffer at lower temperatures, enabling the purification of the target protein from cell lysates. Through exploration of phase separation temperatures, 30 °C was determined to be the optimal purification temperature. Subsequently, three enzymes of different molecular sizes (lipase EstA, endoglucanase BcsZ, and endoglucanase EglS) demonstrated the versatility of this condensate-based purification method. Furthermore, the specific activity and purification efficiency of the purified enzymes were comparable to those of enzymes purified by conventional affinity chromatography. This research contributes to the introduction of condensates into protein purification applications, offering potential support for the large-scale production and purification of functional proteins.