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Pilot-scale extraction of an intracellular recombinant cutinase from E-coli cell homogenate using a thermoseparating aqueous two-phase system
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2003 (English)In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 103, no 2, 165-181 p.Article in journal (Refereed) Published
Abstract [en]

A thermoseparating aqueous two-phase system for extraction of a recombinant cutinase fusion protein from Escherichia coli homogenate has been scaled up to pilot scale. The target protein ZZ-cutinase-(WP)(4) was produced in a fed batch process at 500 1 to a concentration of 12% of the total protein and at a cell concentration of 19.7 g l(-1). After harvest and high-pressure homogenisation a first extraction step was performed in an EO50PO50 (50% (w/w) ethylene oxide and 50% (w/w) propylene oxide) thermopolymer/amylopectin rich Waxy barley starch system. The (WP)4 tag was used for enhanced target protein partitioning to the EO50PO50 phase while the cell debris was collected in the starch phase. A second extraction step followed where the recovered EO50PO50 phase from the first step was supplemented with a non-ionic detergent (C12-18EO5) and heated to the cloud point (CP) temperature (45 degreesC). One polymer-rich liquid phase and one almost pure aqueous phase were formed. The target protein could be obtained in a water phase after the thermal phase separation at a total recovery over the extraction steps of 71% and a purification factor of 2.5. We were able to demonstrate that a disk-stack centrifugal separator could be adapted for rapid separation of both primary and thermoseparated phase systems.

Place, publisher, year, edition, pages
2003. Vol. 103, no 2, 165-181 p.
Keyword [en]
aqueous two-phase system, disk-stack separator, extraction, thermoseparation, cutinase, Escherichia coli, propylene oxide copolymers, induced phase-separation, protein-peptide fusions, bacteriophage-lambda, enzyme-purification, temperature, polymer, galactosidase, behavior
URN: urn:nbn:se:kth:diva-22626DOI: 10.1016/s0168-1656(03)00104-4ISI: 000183825000008OAI: diva2:341324
QC 20100525Available from: 2010-08-10 Created: 2010-08-10Bibliographically approved

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