A novel two-step extraction method with detergent/polymer systems for primary recovery of the fusion protein endoglucanase I-hydrophobin I.
(2002) In Biochimica et Biophysica Acta 1569(1-3). p.139-150- 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... (More)
- 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. (Less)
Please use this url to cite or link to this publication:
https://lup.lub.lu.se/record/107126
- author
- Collén, Anna LU ; Persson, Josefine ; Linder, Markus ; Nakari-Setälä, Tiina ; Penttilä, Merja ; Tjerneld, Folke LU and Sivars, Ulf
- organization
- publishing date
- 2002
- type
- Contribution to journal
- publication status
- published
- subject
- keywords
- Non-U.S. Gov't, Support, Polymers, Recombinant Fusion Proteins : isolation & purification, Micelles, Fungal Proteins : isolation & purification, Fungal Proteins : chemistry, Fermentation, Countercurrent Distribution : methods, Detergents, Trichoderma : metabolism, Temperature, Comparative Study, Cellulase : isolation & purification, Cellulase : chemistry
- in
- Biochimica et Biophysica Acta
- volume
- 1569
- issue
- 1-3
- pages
- 139 - 150
- publisher
- Elsevier
- external identifiers
-
- wos:000174692400018
- scopus:0037081755
- ISSN
- 0006-3002
- DOI
- 10.1016/S0304-4165(01)00244-6
- language
- English
- LU publication?
- yes
- id
- b3fc356e-0f7d-49d5-b977-8ceeaa820353 (old id 107126)
- date added to LUP
- 2016-04-01 16:18:32
- date last changed
- 2022-04-15 03:37:31
@article{b3fc356e-0f7d-49d5-b977-8ceeaa820353, 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 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.}}, author = {{Collén, Anna and Persson, Josefine and Linder, Markus and Nakari-Setälä, Tiina and Penttilä, Merja and Tjerneld, Folke and Sivars, Ulf}}, issn = {{0006-3002}}, keywords = {{Non-U.S. Gov't; Support; Polymers; Recombinant Fusion Proteins : isolation & purification; Micelles; Fungal Proteins : isolation & purification; Fungal Proteins : chemistry; Fermentation; Countercurrent Distribution : methods; Detergents; Trichoderma : metabolism; Temperature; Comparative Study; Cellulase : isolation & purification; Cellulase : chemistry}}, language = {{eng}}, number = {{1-3}}, pages = {{139--150}}, publisher = {{Elsevier}}, series = {{Biochimica et Biophysica Acta}}, title = {{A novel two-step extraction method with detergent/polymer systems for primary recovery of the fusion protein endoglucanase I-hydrophobin I.}}, url = {{http://dx.doi.org/10.1016/S0304-4165(01)00244-6}}, doi = {{10.1016/S0304-4165(01)00244-6}}, volume = {{1569}}, year = {{2002}}, }