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Negatively charged purification tags for selective anion-exchange recovery
KTH, Superseded Departments, Biotechnology.ORCID iD: 0000-0003-0140-419X
KTH, Superseded Departments, Biotechnology.ORCID iD: 0000-0002-5391-600X
KTH, Superseded Departments, Biotechnology.ORCID iD: 0000-0001-8993-048X
KTH, Superseded Departments, Biotechnology.ORCID iD: 0000-0003-0605-8417
2004 (English)In: Protein Engineering Design & Selection, ISSN 1741-0126, E-ISSN 1741-0134, Vol. 17, no 11, 779-786 p.Article in journal (Refereed) Published
Abstract [en]

 A novel strategy for the highly selective purification of recombinant fusion proteins using negatively charged protein domains, which were constructed by protein design, is described. A triple alpha-helical domain of 58 amino acids was used as scaffold. Far-ultraviolet circular dichroism measurements showed that the designed domains had very low alpha-helicity in a low-conductivity environment in contrast to the scaffold. The secondary structure could be induced by adding salt, giving a structure comparable to the parental molecule. Further studies showed that the new domains were able to bind to an anion exchanger even at pH values down to 5 and 6. Gene fusions between one of the designed domains and different target proteins, such as green fluorescent protein (GFP), maltose binding protein (MBP) and firefly luciferase, were also constructed. These gene products could be efficiently purified from whole cell lysates at pH 6 using anion-exchange chromatography.

Place, publisher, year, edition, pages
2004. Vol. 17, no 11, 779-786 p.
Keyword [en]
ion-exchange chromatography, protein A, protein design, Z domain
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:kth:diva-8272DOI: 10.1093/protein/gzh092ISI: 000227133500003Scopus ID: 2-s2.0-14644444571OAI: oai:DiVA.org:kth-8272DiVA: diva2:13550
Note
QC 20100923 QC 20110915Available from: 2005-11-02 Created: 2005-11-02 Last updated: 2017-12-14Bibliographically approved
In thesis
1. Strategies for facilitated protein recovery after recombinant production in Escherichia coli
Open this publication in new window or tab >>Strategies for facilitated protein recovery after recombinant production in Escherichia coli
2005 (English)Doctoral thesis, comprehensive summary (Other scientific)
Abstract [en]

The successful genomic era has resulted in a great demand for efficient production and purification of proteins. The main objective of the work described in this thesis was to develop methods to facilitate recovery of target proteins after recombinant production in Escherichia coli.

A positively charged purification tag, Zbasic, has previously been constructed by protein design of a compact three-helix bundle domain, Z. The charged domain was investigated for general use as a fusion partner. All target proteins investigated could be selectively captured by ion-exchange chromatography under conditions excluding adsorption of the majority of Escherichia coli host proteins. A single cation-exchange chromatography step at physiological pH was sufficient to provide Zbasic fusion proteins of high purity close to homogeneity. Moreover, efficient isolation directly from unclarified Escherichia coli homogenates could also be accomplished using an expanded bed mode. Since the intended use of a recombinant protein sometimes requires removal of the purification tag, a strategy for efficient release of the Zbasic moiety using an immobilised protease was developed. The protease columns were reusable without any measurable decrease in activity. Moreover, subsequent removal of the released tag, Zbasic, was effected by adsorption to a second cation-exchanger.

Using a similar strategy, a purification tag with a negatively charged surface, denoted Zacid, was constructed and thoroughly characterised. Contrary to Zbasic, the negatively charged Zacid was highly unstructured in a low conductivity environment. Despite this, all Zacid fusion proteins investigated could be efficiently purified from whole cell lysates using anion-exchange chromatography

Synthesis of polypeptides occurs readily in Escherichia coli providing large amounts of protein in cells of this type, albeit often one finds the recombinant proteins sequestered in inclusion bodies. Therefore, a high throughput method for screening of protein expression was developed. Levels of both soluble and precipitated protein could simultaneously be assessed in vivo by the use of a flow cytometer.

The positively charged domain, Zbasic, was shown also to be selective under denaturing conditions, providing the possibility to purify proteins solubilised from inclusion bodies. Finally, a flexible process for solid-phase refolding was developed, using Zbasic as a reversible linker to the cation-exchanger resin.

Place, publisher, year, edition, pages
Stockholm: KTH, 2005. 91 p.
Keyword
ion-exchange chromatography, protein A, Z, Zbasic, Zacid, fusion protein, proteolytic cleavage, immobilised protease, flow cytometry, inclusion bodies, solid-phase refolding
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-471 (URN)91-7178-176-5 (ISBN)
Public defence
2005-11-18, Sal FR4, AlbaNova, Roslagstullsbacken 21, Stockholm, 10:15
Opponent
Supervisors
Note
QC 20101020Available from: 2005-11-02 Created: 2005-11-02 Last updated: 2010-10-20Bibliographically approved

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Hedhammar, MyGräslund, TorbjörnUhlén, MathiasHober, Sophia

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