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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

Publiceringsår: 2002
Språk: Engelska
Sidor: 55-62
Publikation/Tidskrift/Serie: Journal of Chromatography A
Volym: 943
Nummer: 1
Dokumenttyp: Artikel i tidskrift
Förlag: Elsevier


Endoglucanases (EGI) (endo-1,4-β-d-glucan-4-glucanohydrolase, EC, 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.


  • Biological Sciences
  • 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


  • ISSN: 0021-9673

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