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Two-step recovery process for tryptophan tagged cutinase: Interfacing aqueous two-phase extraction and hydrophobic interaction chromatography
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2005 (English)In: Journal of Chromatography A, ISSN 0021-9673, E-ISSN 1873-3778, Vol. 1075, no 02-jan, 33-41 p.Article in journal (Refereed) Published
Abstract [en]

In this work, the interfacing of a poly(ethylene glycol) (PEG)-phosphate aqueous two-phase system with hydrophobic interaction chromatography (HIC) for primary recovery of an intracellular protein was evaluated. As a model protein, a recombinant cutinase furnished with a tryptophan-proline (WP) peptide tag was used and produced intracellularly in Escherichia coli (E. coli). E. coli cell homogenate was partitioned in a two-phase system and the top phase yield, concentration and purity of the tagged ZZ-cutinase-(WP)(4) was evaluated as function of polymer sizes, system pH and phase volume ratio. The partition behaviour of cell debris, total protein and endotoxin was also monitored. In the HIC part, the chromatographic yield and purity was investigated with respect to ligand hydrophobicity, dilution of loaded top phase and elution conditions. Based on the results, a recovery process was demonstrated where a PEG 1500-K-Na phosphate salt aqueous two-phase system was interfaced with a HIC column. The interfacing was facilitated by the Trp-tagged peptide. The tagged ZZ-cutinase-(WP)4 was obtained in a PEG-free phase and purified to > 95% purity according to silver stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels with a total yield of 83% during the two-step recovery process. © 2005 Elsevier B.V. All rights reserved.

Place, publisher, year, edition, pages
2005. Vol. 1075, no 02-jan, 33-41 p.
Keyword [en]
aqueous two-phase system, hydrophobic interaction chromatography, extraction, cutinase, Escherichia coli, escherichia-coli, protein-a, lactate-dehydrogenase, beta-galactosidase, phase-separation, systems, purification, identification, partition
URN: urn:nbn:se:kth:diva-14793DOI: 10.1016/j.chroma.2005.03.054ISI: 000229550800003ScopusID: 2-s2.0-19444361831OAI: diva2:332834
QC 20100525Available from: 2010-08-05 Created: 2010-08-05Bibliographically approved

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Veide, Andres
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