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Enzymatic cleavage of fusion proteins using immobilised protease 3C
KTH, School of Biotechnology (BIO), Proteomics.ORCID iD: 0000-0003-0140-419X
University of Southern Denmark, Department for Biochemistry and Molecular Biology.
KTH, School of Biotechnology (BIO), Proteomics.ORCID iD: 0000-0003-0605-8417
2006 (English)In: Protein Expression and Purification, ISSN 1046-5928, E-ISSN 1096-0279, Vol. 47, no 2, 422-426 p.Article in journal (Refereed) Published
Abstract [en]

A strategy for efficient cleavage of fusion proteins using an immobilised protease has been developed. Protease 3C from coxsackie virus was recombinantly produced in Escherichia coli and covalently immobilised onto a solid support. Thereafter, Z(basic) tagged fusion proteins, with a specific cleavage sequence between the domains, were flown through the proteolytic column and circulated until complete cleavage. Subsequently, the processed protein solution was applied on a cation exchanger. Thereby, removal of the released, positively charged fusion tag, Z(basic), was done by adsorption to the matrix while the target proteins were recovered in the flow through. Interestingly, the columns were shown to be reusable without any measurable decrease in activity. Moreover, after storage in 4 degrees C for two months the activity was almost unaffected.

Place, publisher, year, edition, pages
2006. Vol. 47, no 2, 422-426 p.
Keyword [en]
immobilised protease, Z(basic), protease 3C, cleavage, purification, efficient
National Category
Biochemistry and Molecular Biology
URN: urn:nbn:se:kth:diva-15745DOI: 10.1016/j.pep.2006.01.003ISI: 000238277000011ScopusID: 2-s2.0-33646541425OAI: diva2:333787
QC 20100525Available from: 2010-08-05 Created: 2010-08-05 Last updated: 2010-10-20Bibliographically 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.
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
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
QC 20101020Available from: 2005-11-02 Created: 2005-11-02 Last updated: 2010-10-20Bibliographically approved

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