Advanced

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)
Please use this url to cite or link to this publication:
author
, et al. (More)
(Less)
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
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
2007-07-04 14:54:19
date last changed
2017-01-01 04:40:39
@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},
  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},
  volume       = {78},
  year         = {2002},
}