A novel two-step extraction method with detergent/polymer systems for primary recovery of the fusion protein endoglucanase I-hydrophobin I.
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 degrees 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.
Collén
Anna
A
Depqartment of Biochemistry, Center for Chemistry and Chemical Engineering, Lund University, Sweden.
Persson
Josefine
J
Linder
Markus
M
Nakari-Setälä
Tiina
T
Penttilä
Merja
M
Tjerneld
Folke
F
Sivars
Ulf
U
D002480:Cellulase / Q000737:chemistry / Q000302:isolation & purification*; D043366:Cellulose 1,4-beta-Cellobiosidase; D003377:Countercurrent Distribution / Q000379:methods*; D003902:Detergents; D005285:Fermentation; D005656:Fungal Proteins / Q000737:chemistry / Q000302:isolation & purification*; D008823:Micelles; D011108:Polymers; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification*; D013696:Temperature; D014242:Trichoderma / Q000378:metabolism*; D014867:Water
D003902:Detergents; D005656:Fungal Proteins; D008823:Micelles; D011108:Polymers; D011993:Recombinant Fusion Proteins; D014867:Water; D002480:Cellulase; D043366:Cellulose 1,4-beta-Cellobiosidase