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Primary recovery of a genetically engineered Trichoderma reesei endoglucanase I (Cel 7B) fusion protein in cloud point extraction systems.

Collén, Anna LU ; Selber, Klaus ; Hyytiä, Teppo ; Persson, Josefine ; Nakari-Setlä, Tiina ; Bailey, Michael ; Fagerström, Richard ; Kula, Maria-Regina ; Penttilä, Merja and Stålbrand, Henrik LU , et al. (2002) In Biotechnology and Bioengineering 78(4). p.385-394
Abstract
Here we present data to demonstrate how partitioning of a hydrophilic enzyme can be directed to the hydrophobic detergent-enriched phase of an aqueous two-phase system by addition of short stretches of amino acid residues to the protein molecule. The target enzyme was the industrially important endoglucanase I, EGI (endo-1,4-beta-D-glucan-4-glucanohydrolase, EC 3.2.1.4, Cel7B) of Trichoderma reesei. We investigated the partitioning of three EGI variants containing various C-terminal peptide extensions including Trp-Pro motifs of different lengths and localizations. Additionally, a recently developed system composed of the thermoseparating copolymer HM-EOPO was utilized to study the effects of fusion tags. The addition of peptides... (More)
Here we present data to demonstrate how partitioning of a hydrophilic enzyme can be directed to the hydrophobic detergent-enriched phase of an aqueous two-phase system by addition of short stretches of amino acid residues to the protein molecule. The target enzyme was the industrially important endoglucanase I, EGI (endo-1,4-beta-D-glucan-4-glucanohydrolase, EC 3.2.1.4, Cel7B) of Trichoderma reesei. We investigated the partitioning of three EGI variants containing various C-terminal peptide extensions including Trp-Pro motifs of different lengths and localizations. Additionally, a recently developed system composed of the thermoseparating copolymer HM-EOPO was utilized to study the effects of fusion tags. The addition of peptides containing tryptohan residues enhanced the partitioning of EGI to the HM-EOPO-rich phase. The system composed of a nonionic detergent (Agrimul NRE1205) resulted in the highest partition coefficient (K = 31) and yield (90%) with the construct EGI(core-P5)(WP)(4) containing (Trp-Pro)(4) after a short linker stretch. A recombinant strain of T. reesei Rut-C30 for large-scale production was constructed in which the fusion protein EGI(core-P5)(WP)(4) was expressed from the strong promoter of the cellulase gene cbh1. The fusion protein was successfully expressed and secreted from the fungus during shake-flask cultivations. Cultivation in a 28-L bioreactor however, revealed that the fusion protein is sensitive to proteases. Consequently, only low production levels were obtained in large-scale production trials. (Less)
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organization
publishing date
type
Contribution to journal
publication status
published
subject
in
Biotechnology and Bioengineering
volume
78
issue
4
pages
385 - 394
publisher
John Wiley & Sons Inc.
external identifiers
  • pmid:11948445
  • wos:000175353900004
  • scopus:0037141601
ISSN
1097-0290
DOI
10.1002/bit.10232
language
English
LU publication?
yes
id
d4d6d1cc-c8f4-40db-ae4b-de42a93c5b2e (old id 107598)
date added to LUP
2016-04-01 11:55:53
date last changed
2022-02-25 23:24:28
@article{d4d6d1cc-c8f4-40db-ae4b-de42a93c5b2e,
  abstract     = {{Here we present data to demonstrate how partitioning of a hydrophilic enzyme can be directed to the hydrophobic detergent-enriched phase of an aqueous two-phase system by addition of short stretches of amino acid residues to the protein molecule. The target enzyme was the industrially important endoglucanase I, EGI (endo-1,4-beta-D-glucan-4-glucanohydrolase, EC 3.2.1.4, Cel7B) of Trichoderma reesei. We investigated the partitioning of three EGI variants containing various C-terminal peptide extensions including Trp-Pro motifs of different lengths and localizations. Additionally, a recently developed system composed of the thermoseparating copolymer HM-EOPO was utilized to study the effects of fusion tags. The addition of peptides containing tryptohan residues enhanced the partitioning of EGI to the HM-EOPO-rich phase. The system composed of a nonionic detergent (Agrimul NRE1205) resulted in the highest partition coefficient (K = 31) and yield (90%) with the construct EGI(core-P5)(WP)(4) containing (Trp-Pro)(4) after a short linker stretch. A recombinant strain of T. reesei Rut-C30 for large-scale production was constructed in which the fusion protein EGI(core-P5)(WP)(4) was expressed from the strong promoter of the cellulase gene cbh1. The fusion protein was successfully expressed and secreted from the fungus during shake-flask cultivations. Cultivation in a 28-L bioreactor however, revealed that the fusion protein is sensitive to proteases. Consequently, only low production levels were obtained in large-scale production trials.}},
  author       = {{Collén, Anna and Selber, Klaus and Hyytiä, Teppo and Persson, Josefine and Nakari-Setlä, Tiina and Bailey, Michael and Fagerström, Richard and Kula, Maria-Regina and Penttilä, Merja and Stålbrand, Henrik and Tjerneld, Folke}},
  issn         = {{1097-0290}},
  language     = {{eng}},
  number       = {{4}},
  pages        = {{385--394}},
  publisher    = {{John Wiley & Sons Inc.}},
  series       = {{Biotechnology and Bioengineering}},
  title        = {{Primary recovery of a genetically engineered Trichoderma reesei endoglucanase I (Cel 7B) fusion protein in cloud point extraction systems.}},
  url          = {{http://dx.doi.org/10.1002/bit.10232}},
  doi          = {{10.1002/bit.10232}},
  volume       = {{78}},
  year         = {{2002}},
}