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

Collén, Anna LU ; Persson, Josefine; Linder, Markus; Nakari-Setälä, Tiina; Penttilä, Merja; Tjerneld, Folke LU and Sivars, Ulf (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)
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author
organization
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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
2007-07-04 14:57:57
date last changed
2017-07-30 04:35:38
@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},
  keyword      = {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},
  volume       = {1569},
  year         = {2002},
}