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

• Published in: Biochimica et biophysica acta Vol. 1569; no. 1; pp. 139 - 150
• Main Authors: Collén, Anna, Persson, Josefine, Linder, Markus, Nakari-Setälä, Tiina, Penttilä, Merja, Tjerneld, Folke, Sivars, Ulf
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 15.01.2002
• Subjects: Aqueous two-phase system; Biologi; Biological Sciences; Cellulase - chemistry; Cellulase - isolation & purification; Cellulose 1,4-beta-Cellobiosidase; Comparative Study; Countercurrent Distribution - methods; Detergent removal; Detergents; Extraction; Fermentation; Fungal Proteins - chemistry; Fungal Proteins - isolation & purification; Fusion protein; Hydrophobin; Micelles; Natural Sciences; Naturvetenskap; Non-U.S. Gov't; Polymers; Protein purification; Recombinant Fusion Proteins - isolation & purification; Support; Temperature; Trichoderma - metabolism; Water; Extraction; Hydrophobin; Fusion protein; Protein purification; Aqueous two-phase system; Detergent removal
• ISSN: 0304-4165; 0006-3002; 1878-2434; 1872-8006
• DOI: 10.1016/S0304-4165(01)00244-6

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.