A novel two-step extraction method with detergent/polymer systems for primary recovery of the fusion protein endoglucanase I–hydrophobin I

Abstract

Extraction systems for hydrophobically tagged proteins have been developed based on phase separation in aqueous solutions of non-ionic detergents and polymers. The systems have earlier only been applied for separation of membrane proteins. Here, we examine the partitioning and purification of the amphiphilic fusion protein endoglucanase I core–hydrophobin I (EGI core–HFBI) from culture filtrate originating from a Trichoderma reesei fermentation. The micelle extraction system was formed by mixing the non-ionic detergent Triton X-114 or Triton X-100 with the hydroxypropyl starch polymer, Reppal PES100. The detergent/polymer aqueous two-phase systems resulted in both better separation characteristics and increased robustness compared to cloud point extraction in a Triton X-114/water system. Separation and robustness were characterized for the parameters: temperature, protein and salt additions. In the Triton X-114/Reppal PES100 detergent/polymer system EGI core–HFBI strongly partitioned into the micelle-rich phase with a partition coefficient ( K) of 15 and was separated from hydrophilic proteins, which preferably partitioned to the polymer phase. After the primary recovery step, EGI core–HFBI was quantitatively back-extracted ( K EGIcore–HFBI=150, yield=99%) into a water phase. In this second step, ethylene oxide–propylene oxide (EOPO) copolymers were added to the micelle-rich phase and temperature-induced phase separation at 55°C was performed. Total recovery of EGI core–HFBI after the two separation steps was 90% with a volume reduction of six times. For thermolabile proteins, the back-extraction temperature could be decreased to room temperature by using a hydrophobically modified EOPO copolymer, with slightly lower yield. The addition of thermoseparating co-polymer is a novel approach to remove detergent and effectively releases the fusion protein EGI core–HFBI into a water phase.