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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
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2002 (English)In: Journal of Chromatography A, ISSN 0021-9673, E-ISSN 1873-3778, Vol. 943, no 1, 55-62 p.Article in journal (Refereed) Published
Abstract [en]

Endoglucanases (EGI) (endo-1,4-beta-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 [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.

Place, publisher, year, edition, pages
2002. Vol. 943, no 1, 55-62 p.
Keyword [en]
Trichoderma reesei, aqueous two-phase systems, endoglucanases, enzymes, proteins, poly(ethylene glycol), sodium phosphate, potassium phosphate, phosphates, 2-phase systems, gene, purification, domain
URN: urn:nbn:se:kth:diva-21232ISI: 000173094700006OAI: diva2:339930
QC 20100525Available from: 2010-08-10 Created: 2010-08-10Bibliographically approved

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Veide, Andres
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