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Extraction of endoglucanase I (Cel7B) fusion proteins from Trichoderma reesei culture filtrate in a poly(ethylene glycol)-phosphate aqueous two-phase system

Collén, Anna LU ; Penttilä, Merja; Stålbrand, Henrik LU ; Tjerneld, Folke LU and Veide, Andres (2002) In Journal of Chromatography A 943(1). p.55-62
Abstract
Endoglucanases (EGI) (endo-1,4-β-d-glucan-4-glucanohydrolase, EC 3.2.1.4, Cel7B) of Trichoderma reesei are industrially important enzymes. Thus, there is a great need for development of a primary recovery method suitable for large-scale utilization. In this study we present a concept applicable for large-scale purification of an EGI fusion protein by one-step extraction in a poly(ethylene glycol) PEG-sodium/potassium phosphate aqueous two-phase system. EGI is a two-module enzyme composed of an N-terminal catalytic module and a C-terminal cellulose binding module (CBM) separated by a glycosylated linker region. Partitioning of six different EGI constructs, containing the C-terminal extensions (WP)2, (WP)4 or the amphiphilic protein... (More)
Endoglucanases (EGI) (endo-1,4-β-d-glucan-4-glucanohydrolase, EC 3.2.1.4, Cel7B) of Trichoderma reesei are industrially important enzymes. Thus, there is a great need for development of a primary recovery method suitable for large-scale utilization. In this study we present a concept applicable for large-scale purification of an EGI fusion protein by one-step extraction in a poly(ethylene glycol) PEG-sodium/potassium phosphate aqueous two-phase system. EGI is a two-module enzyme composed of an N-terminal catalytic module and a C-terminal cellulose binding module (CBM) separated by a glycosylated linker region. Partitioning of six different EGI constructs, containing the C-terminal extensions (WP)2, (WP)4 or the amphiphilic protein hydrophobin I (HFB) of T. reesei instead of the CBM were studied to evaluate if any of the fusions could improve the partition coefficient sufficiently to be suitable for large-scale production. All constructs showed improved partitioning in comparison to full length EGI. The (WP)4 extensions resulted in 26- to 60-fold improvement of partition coefficient. Consequently, a relative minor change in amino acid sequence on the two-module protein EGI improved the partition coefficient significantly in the PEG 4000-sodium/potassium phosphate system. The addition of HFBI to EGI clearly enhanced the partition coefficient (K=1.2) in comparison to full-length EGI (K=0.035). Partitioning of the construct with (WP)4 fused to the catalytic module and a short sequence of the linker [EGIcore-P5(WP)4] resulted in the highest partition coefficient (K=54) and a yield of 98% in the PEG phase. Gel electrophoresis showed that the construct with the (WP)4 tag attached after a penta-proline linker could be purified from the other bulk proteins by only a single-step separation in the PEG 4000-sodium/potassium phosphate system. This is a major improvement in comparison with the previously studied model (ethylene oxide-propylene oxide)-dextran system. Hence, this construct will be suitable for further optimization of the extraction of the enzyme in a PEG 4000-sodium/potassium phosphate system from culture filtrate. (Less)
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keywords
Polyethylene Glycols/*chemistry, Polyacrylamide Gel, Phosphates/*chemistry, Recombinant Fusion Proteins/isolation & purification/metabolism, Support, Non-U.S. Gov't, Water/chemistry, Trichoderma/chemistry/*enzymology, Electrophoresis, Cellulase/*isolation & purification/metabolism
in
Journal of Chromatography A
volume
943
issue
1
pages
55 - 62
publisher
Elsevier
external identifiers
  • wos:000173094700006
  • pmid:11820281
  • scopus:0037059484
ISSN
0021-9673
DOI
10.1016/S0021-9673(01)01433-9
language
English
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yes
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cb669401-dcba-421e-9649-2a40c1b5c461 (old id 124935)
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http://www.sciencedirect.com/science?_ob=MImg&_imagekey=B6TG8-44GHVVJ-B-D&_cdi=5248&_orig=search&_coverDate=01%2F11%2F2002&_qd=1&_sk=990569998&view=c&wchp=dGLbVzb-zSkzk&_acct=C000041498&_version=1&_userid=745831&md5=ba44424b78e8a62d4bf2b55992796f4c&ie=f.p
date added to LUP
2007-07-04 15:42:40
date last changed
2017-07-30 04:39:40
@article{cb669401-dcba-421e-9649-2a40c1b5c461,
  abstract     = {Endoglucanases (EGI) (endo-1,4-β-d-glucan-4-glucanohydrolase, EC 3.2.1.4, Cel7B) of Trichoderma reesei are industrially important enzymes. Thus, there is a great need for development of a primary recovery method suitable for large-scale utilization. In this study we present a concept applicable for large-scale purification of an EGI fusion protein by one-step extraction in a poly(ethylene glycol) PEG-sodium/potassium phosphate aqueous two-phase system. EGI is a two-module enzyme composed of an N-terminal catalytic module and a C-terminal cellulose binding module (CBM) separated by a glycosylated linker region. Partitioning of six different EGI constructs, containing the C-terminal extensions (WP)2, (WP)4 or the amphiphilic protein hydrophobin I (HFB) of T. reesei instead of the CBM were studied to evaluate if any of the fusions could improve the partition coefficient sufficiently to be suitable for large-scale production. All constructs showed improved partitioning in comparison to full length EGI. The (WP)4 extensions resulted in 26- to 60-fold improvement of partition coefficient. Consequently, a relative minor change in amino acid sequence on the two-module protein EGI improved the partition coefficient significantly in the PEG 4000-sodium/potassium phosphate system. The addition of HFBI to EGI clearly enhanced the partition coefficient (K=1.2) in comparison to full-length EGI (K=0.035). Partitioning of the construct with (WP)4 fused to the catalytic module and a short sequence of the linker [EGIcore-P5(WP)4] resulted in the highest partition coefficient (K=54) and a yield of 98% in the PEG phase. Gel electrophoresis showed that the construct with the (WP)4 tag attached after a penta-proline linker could be purified from the other bulk proteins by only a single-step separation in the PEG 4000-sodium/potassium phosphate system. This is a major improvement in comparison with the previously studied model (ethylene oxide-propylene oxide)-dextran system. Hence, this construct will be suitable for further optimization of the extraction of the enzyme in a PEG 4000-sodium/potassium phosphate system from culture filtrate.},
  author       = {Collén, Anna and Penttilä, Merja and Stålbrand, Henrik and Tjerneld, Folke and Veide, Andres},
  issn         = {0021-9673},
  keyword      = {Polyethylene Glycols/*chemistry,Polyacrylamide Gel,Phosphates/*chemistry,Recombinant Fusion Proteins/isolation & purification/metabolism,Support,Non-U.S. Gov't,Water/chemistry,Trichoderma/chemistry/*enzymology,Electrophoresis,Cellulase/*isolation & purification/metabolism},
  language     = {eng},
  number       = {1},
  pages        = {55--62},
  publisher    = {Elsevier},
  series       = {Journal of Chromatography A},
  title        = {Extraction of endoglucanase I (Cel7B) fusion proteins from Trichoderma reesei culture filtrate in a poly(ethylene glycol)-phosphate aqueous two-phase system},
  url          = {http://dx.doi.org/10.1016/S0021-9673(01)01433-9},
  volume       = {943},
  year         = {2002},
}