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Integrated strategy for selective expanded bed ion-exchange adsorption and site-specific protein processing using gene fusion technology
KTH, Superseded Departments, Biochemistry and Biotechnology.ORCID iD: 0000-0002-5391-600X
KTH, Superseded Departments, Biochemistry and Biotechnology.ORCID iD: 0000-0003-0140-419X
KTH, Superseded Departments, Biochemistry and Biotechnology.ORCID iD: 0000-0001-8993-048X
KTH, Superseded Departments, Biochemistry and Biotechnology.ORCID iD: 0000-0003-4214-6991
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2002 (English)In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 96, no 1, p. 93-102Article in journal (Refereed) Published
Abstract [en]

The highly charged domain Z(basic) can be used as a fusion partner to enhance adsorption of target proteins to cation exchanging resins at high pH-values. In this paper, we describe a strategy for purification of target proteins fused to Z(basic) at a constant physiological pH using cation exchange chromatography in an expanded bed mode. We show that two proteins, Klenow DNA polymerase and the viral protease 3C, can be efficiently purified from unclarified Escherichia coli homogenates in a single step with a selectivity analogous to what is normally achieved by affinity chromatography. The strategy also includes an integrated site-specific removal of the Z(basic) purification handle to yield a free target protein.

Place, publisher, year, edition, pages
2002. Vol. 96, no 1, p. 93-102
Keywords [en]
protein A, Z(basic), ion-exchange chromatography, expanded bed adsorption, DNA-POLYMERASE-I, ESCHERICHIA-COLI, PURIFICATION, RECOVERY, COXSACKIEVIRUS-B3, CHROMATOGRAPHY, EXPRESSION, EFFICIENT, CELLS
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:kth:diva-13276DOI: 10.1016/S0168-1656(02)00040-8ISI: 000176318300010OAI: oai:DiVA.org:kth-13276DiVA, id: diva2:323049
Note
QC 20100609Available from: 2010-06-09 Created: 2010-06-09 Last updated: 2017-12-12Bibliographically approved
In thesis
1. Protein Engineering by Directed Evolution and Rational Design
Open this publication in new window or tab >>Protein Engineering by Directed Evolution and Rational Design
2001 (English)Doctoral thesis, comprehensive summary (Other scientific)
Place, publisher, year, edition, pages
Stockholm: KTH, 2001. p. 87
Keywords
ion-exchange chromatography, subtilisin, Klenow DNA polymerase, Taq DNA polymerase, directed evolution, rational design, charge engineering, phosphonylating inhibitor, phage display, circular dichroism, protein A
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-3171 (URN)91-7283-102-2 (ISBN)
Public defence
2001-06-01, 00:00
Note
QC 20100609Available from: 2001-05-31 Created: 2001-05-31 Last updated: 2010-06-09Bibliographically approved
2. 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. p. 91
Keywords
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: 2018-01-13Bibliographically approved

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Gräslund, TorbjörnHedhammar, MyUhlén, MathiasNygren, Per-ÅkeHober, Sophia

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