Extraction of endoglucanase I (Cel7B) fusion proteins from Trichoderma reesei culture filtrate in a poly(ethylene glycol)-phosphate aqueous two-phase system
(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)
Please use this url to cite or link to this publication:
https://lup.lub.lu.se/record/124935
- author
- Collén, Anna LU ; Penttilä, Merja ; Stålbrand, Henrik LU ; Tjerneld, Folke LU and Veide, Andres
- organization
- publishing date
- 2002
- type
- Contribution to journal
- publication status
- published
- subject
- 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
- LU publication?
- yes
- id
- cb669401-dcba-421e-9649-2a40c1b5c461 (old id 124935)
- alternative location
- 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
- 2016-04-01 16:39:33
- date last changed
- 2022-01-28 21:12:17
@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}}, 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}}, 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}}, doi = {{10.1016/S0021-9673(01)01433-9}}, volume = {{943}}, year = {{2002}}, }