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

Anna Collén, Merja Penttilä, Henrik Stålbrand, Folke Tjerneld, Andres Veide

Research output: Contribution to journalArticleScientificpeer-review

9 Citations (Scopus)

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 [EGI core-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.

Original languageEnglish
Pages (from-to)55-62
Number of pages8
JournalJournal of Chromatography A
Volume943
Issue number1
DOIs
Publication statusPublished - 11 Jan 2002
MoE publication typeA1 Journal article-refereed

Fingerprint

Cellulases
Trichoderma
Ethylene Glycol
Polyethylene glycols
Fusion reactions
Phosphates
Sodium
Cellulose
Proteins
Enzymes
Ethylene Oxide
Rubiaceae
Cellulase
Dextrans
Proline
Electrophoresis
Amino Acid Sequence
Gels
Purification
potassium phosphate

Keywords

  • Aqueous two-phase systems
  • Trichoderma reesei
  • Endoglucanases
  • Enzymes
  • Proteins
  • Sodium phosphate
  • potassium
  • phosphates

Cite this

@article{89768837a0534669bb970c9aaf04061b,
title = "Extraction of endoglucanase I (Cel7B) fusion proteins from Trichoderma reesei culture filtrate in a poly(ethylene glycol)-phosphate aqueous two-phase system",
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 [EGI core-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.",
keywords = "Aqueous two-phase systems, Trichoderma reesei, Endoglucanases, Enzymes, Proteins, Sodium phosphate, potassium, phosphates",
author = "Anna Coll{\'e}n and Merja Penttil{\"a} and Henrik St{\aa}lbrand and Folke Tjerneld and Andres Veide",
year = "2002",
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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; Penttilä, Merja; Stålbrand, Henrik; Tjerneld, Folke; Veide, Andres.

In: Journal of Chromatography A, Vol. 943, No. 1, 11.01.2002, p. 55-62.

Research output: Contribution to journalArticleScientificpeer-review

TY - JOUR

T1 - Extraction of endoglucanase I (Cel7B) fusion proteins from Trichoderma reesei culture filtrate in a poly(ethylene glycol)-phosphate aqueous two-phase system

AU - Collén, Anna

AU - Penttilä, Merja

AU - Stålbrand, Henrik

AU - Tjerneld, Folke

AU - Veide, Andres

PY - 2002/1/11

Y1 - 2002/1/11

N2 - 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 [EGI core-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.

AB - 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 [EGI core-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.

KW - Aqueous two-phase systems

KW - Trichoderma reesei

KW - Endoglucanases

KW - Enzymes

KW - Proteins

KW - Sodium phosphate

KW - potassium

KW - phosphates

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U2 - 10.1016/S0021-9673(01)01433-9

DO - 10.1016/S0021-9673(01)01433-9

M3 - Article

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EP - 62

JO - Journal of Chromatography A

JF - Journal of Chromatography A

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