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A novel two-step ex...
A novel two-step extraction method with detergent/polymer systems for primary recovery of the fusion protein endoglucanase I-hydrophobin I.
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- Collén, Anna (författare)
- Lund University,Lunds universitet,Biokemi och Strukturbiologi,Centrum för Molekylär Proteinvetenskap,Kemiska institutionen,Institutioner vid LTH,Lunds Tekniska Högskola,Biochemistry and Structural Biology,Center for Molecular Protein Science,Department of Chemistry,Departments at LTH,Faculty of Engineering, LTH
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Persson, Josefine (författare)
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Linder, Markus (författare)
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Nakari-Setälä, Tiina (författare)
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Penttilä, Merja (författare)
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- Tjerneld, Folke (författare)
- Lund University,Lunds universitet,Biokemi och Strukturbiologi,Centrum för Molekylär Proteinvetenskap,Kemiska institutionen,Institutioner vid LTH,Lunds Tekniska Högskola,Biochemistry and Structural Biology,Center for Molecular Protein Science,Department of Chemistry,Departments at LTH,Faculty of Engineering, LTH
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Sivars, Ulf (författare)
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(creator_code:org_t)
- 2002
- 2002
- Engelska.
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Ingår i: Biochimica et Biophysica Acta. - 0006-3002. ; 1569:1-3, s. 139-150
- Relaterad länk:
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http://dx.doi.org/10...
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https://lup.lub.lu.s...
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https://doi.org/10.1...
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Abstract
Ämnesord
Stäng
- 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.
Ämnesord
- NATURVETENSKAP -- Biologi (hsv//swe)
- NATURAL SCIENCES -- Biological Sciences (hsv//eng)
Nyckelord
- 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
Publikations- och innehållstyp
- art (ämneskategori)
- ref (ämneskategori)
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