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Hydrophobic Fusion Tags: Implications For Bioseparation and Cellular Expression

Collén, Anna LU (2001)
Abstract
The studies in this thesis have shown that the partitioning of endoglucanase I (EGI, Cel7B) from T. reesei could be significantly improved by relatively minor genetic engineering. By adding short peptides composed of tryptophan and proline residues to EGI, extreme partitioning could be obtained. The site of the tag fusion was shown to be crucial for the efficiency of the tag. Methods suitable for large-scale purification of genetically modified EGI by a single-step extraction in aqueous two-phase systems have been established. The most optimal fusion protein, with respect to partitioning enhancement resulted, however, in impaired production in T. reesei. This was further elucidated and the low production was suggested to be caused by... (More)
The studies in this thesis have shown that the partitioning of endoglucanase I (EGI, Cel7B) from T. reesei could be significantly improved by relatively minor genetic engineering. By adding short peptides composed of tryptophan and proline residues to EGI, extreme partitioning could be obtained. The site of the tag fusion was shown to be crucial for the efficiency of the tag. Methods suitable for large-scale purification of genetically modified EGI by a single-step extraction in aqueous two-phase systems have been established. The most optimal fusion protein, with respect to partitioning enhancement resulted, however, in impaired production in T. reesei. This was further elucidated and the low production was suggested to be caused by several factors such as proteolysis, impaired secretion and possible down-regulation of the promoter due to intracellular accumulation of the hydrophobic fusion protein. At certain stages during growth of the transformant expressing EGIcore-P5(WP)4 slight induction of the gene encoding the ER residual chaperone BIPI was detected.



Furthermore, the amphiphilic protein hydrophobin I was utilized as a fusion tag to direct partitioning in aqueous two-phase systems. A system with improved separation features was evaluated, which is a clear progression from previously used systems both with respect to both robustness and purification properties. Applications towards large-scale purification with this system might be possible in the foreseeable future. Additionally, a novel approach for detergent removal after two-phase extraction in detergent based systems was developed. By addition of thermoseparating polymers, HM-EOPO or EOPO, phase separation could be induced by temperature increase, and thus the fusion protein could be recovered in a water phase. This method is both environmentally benign and displays compatibility with subsequent purification steps and handling of the target protein. (Less)
Please use this url to cite or link to this publication:
author
opponent
  • Prof Archer, David, University of Nottingham, UK
organization
publishing date
type
Thesis
publication status
published
subject
keywords
protein production, secretion, aqueous two-phase systems, Trichoderma reesei, endoglucanase, Biochemistry, Metabolism, Biokemi, metabolism
pages
178 pages
publisher
Department of Biochemistry, Lund University
defense location
Sal A Chemical Centre
defense date
2001-10-12 10:15
external identifiers
  • other:ISRN: LUNKDL/(NKBK-1070)/1-178/2001
ISBN
91-7874-140-8
language
English
LU publication?
yes
id
0e8f3aae-0040-4429-bc8a-28a590938af9 (old id 41896)
date added to LUP
2007-06-20 12:24:11
date last changed
2016-09-19 08:45:12
@phdthesis{0e8f3aae-0040-4429-bc8a-28a590938af9,
  abstract     = {The studies in this thesis have shown that the partitioning of endoglucanase I (EGI, Cel7B) from T. reesei could be significantly improved by relatively minor genetic engineering. By adding short peptides composed of tryptophan and proline residues to EGI, extreme partitioning could be obtained. The site of the tag fusion was shown to be crucial for the efficiency of the tag. Methods suitable for large-scale purification of genetically modified EGI by a single-step extraction in aqueous two-phase systems have been established. The most optimal fusion protein, with respect to partitioning enhancement resulted, however, in impaired production in T. reesei. This was further elucidated and the low production was suggested to be caused by several factors such as proteolysis, impaired secretion and possible down-regulation of the promoter due to intracellular accumulation of the hydrophobic fusion protein. At certain stages during growth of the transformant expressing EGIcore-P5(WP)4 slight induction of the gene encoding the ER residual chaperone BIPI was detected.<br/><br>
<br/><br>
Furthermore, the amphiphilic protein hydrophobin I was utilized as a fusion tag to direct partitioning in aqueous two-phase systems. A system with improved separation features was evaluated, which is a clear progression from previously used systems both with respect to both robustness and purification properties. Applications towards large-scale purification with this system might be possible in the foreseeable future. Additionally, a novel approach for detergent removal after two-phase extraction in detergent based systems was developed. By addition of thermoseparating polymers, HM-EOPO or EOPO, phase separation could be induced by temperature increase, and thus the fusion protein could be recovered in a water phase. This method is both environmentally benign and displays compatibility with subsequent purification steps and handling of the target protein.},
  author       = {Collén, Anna},
  isbn         = {91-7874-140-8},
  keyword      = {protein production,secretion,aqueous two-phase systems,Trichoderma reesei,endoglucanase,Biochemistry,Metabolism,Biokemi,metabolism},
  language     = {eng},
  pages        = {178},
  publisher    = {Department of Biochemistry, Lund University},
  school       = {Lund University},
  title        = {Hydrophobic Fusion Tags: Implications For Bioseparation and Cellular Expression},
  year         = {2001},
}