Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • harvard1
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Single-step recovery and solid-phase refolding of inclusion body proteins using a polycationic purification tag
KTH, School of Biotechnology (BIO), Proteomics.ORCID iD: 0000-0003-0140-419X
KTH, School of Biotechnology (BIO), Proteomics.ORCID iD: 0000-0002-2643-8241
KTH, School of Biotechnology (BIO), Proteomics.ORCID iD: 0000-0002-5391-600X
KTH, School of Biotechnology (BIO), Proteomics.ORCID iD: 0000-0003-0605-8417
2006 (English)In: Biotechnology Journal, ISSN 1860-6768, Vol. 1, 187-196 p.Article in journal (Refereed) Published
Abstract [en]

A strategy for purification of inclusion body-forming proteins is described, in which the positively charged domain Z(basic) is used as a fusion partner for capture of denatured proteins on a cation exchange column. It is shown that the purification tag is selective under denaturing conditions. Furthermore, the new strategy for purification of proteins from inclusion bodies is compared with the commonly used method for purification of His(6)-tagged inclusion body proteins. Finally, the simple and effective means of target protein capture provided by the Z(basic) tag is further successfully explored for solid-phase refolding. This procedure has the inherited advantage of combining purification and refolding in one step and offers the advantage of eluting the concentrated product in a suitable buffer.

Place, publisher, year, edition, pages
2006. Vol. 1, 187-196 p.
Keyword [en]
Zbasic, Ion exchange chromatography, Purification of inclusion body proteins, Solid-phase refolding
Identifiers
URN: urn:nbn:se:kth:diva-12815DOI: 10.1002/biot.200500023PubMedID: 16892247Scopus ID: 2-s2.0-33750601043OAI: oai:DiVA.org:kth-12815DiVA: diva2:319007
Note
QC20100622Available from: 2010-05-12 Created: 2010-05-12 Last updated: 2010-10-20Bibliographically approved
In thesis
1. Interaction engineered three-helix bundle domains for protein recovery and detection
Open this publication in new window or tab >>Interaction engineered three-helix bundle domains for protein recovery and detection
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

HTML clipboard The great advances in DNA technology, e.g. sequencing and recombinant DNA techniques, have given us the genetic information and the tools needed to effectively produce recombinant proteins. Recombinant proteins are valuable means in biotechnological applications and are also emerging as alternatives in therapeutic applications. Traditionally, monoclonal antibodies have been the natural choice for biotechnological and therapeutic applications due to their ability to bind a huge range of different molecules and their natural good affinity. However, the large size of antibodies (150 kDa) limits tissue penetration and the recombinant expression is complicated. Therefore, alternative binders with smaller sizes have been derived from antibodies and alternative scaffolds.

In this thesis, two structurally similar domains, Zbasic and ABDz1, have been used as purification tags in different contexts. They are both three-helical bundles and derived from bacterial surface domains, but share no sequence homology. Furthermore, by redesign of the scaffold used for ABDz1, a molecule intended for drug targeting with extended in-vivo half-life has been engineered. In Papers I and II, the poly-cationic tag Zbasic is explored and evaluated. Paper I describes the successful investigation of Zbasic as a purification handle under denaturating conditions. Moreover, Zbasic is evaluated as an interaction domain in matrixassisted refolding. Two different proteins were successfully refolded using the same setup without individual optimization. In Paper II, Zbasic is further explored as a purification handle under non-native conditions in a multi-parallel setup. In total, 22 proteins with varying characteristics are successfully purified using a multi-parallel protein purification protocol and a robotic system. Without modifications, the system can purify up to 60 proteins without manual handling. Paper I and II clearly demonstrate that Zbasic can be used as an interaction domain in matrix-assisted refolding and that it offers a good alternative to the commonly used His6-tag under denaturating conditions. In paper III, the small bifunctional ABDz1 is selected from a phage display library. Endowed with two different binding interfaces, ABDz1 is capable of binding both the HSA-sepharose and the protein A-derived MabSelect SuRe-matrix. The bifunctionality of the domain is exploited in an orthogonal affinity setup. Three target proteins are successfully purified using the HSA-matrix and the MabSelect SuRe-matrix. Furthermore, the purity of the target proteins is effectively improved by combining the two chromatographic steps. Thus, paper III shows that the small ABDz1 can be used as an effective purification handle and dual affinity tag without target specific optimization. Paper IV describes the selection and affinity maturation of small bispecific drug-targeting molecules. First generation binders against tumor necrosis factor-α are selected using phage display. Thereafter on-cell surface display and flow cytometry is used to select second-generation binders. The binding to tumor necrosis factor-α is improved up to 30 times as compared to the best first generation binder, and a 6-fold improvement of the binding strength was possible with retained HSA affinity. Paper III and IV clearly demonstrate that dual interaction surfaces can successfully be grafted on a small proteinaceous domain, and that the strategy in paper IV can be used for dual selection of bifunctional binders.

Place, publisher, year, edition, pages
Stockholm: KTH, 2010. x, 79 p.
Series
Trita-BIO-Report, ISSN 1654-2312 ; 2010:5
Keyword
ABD, Zbasic, albumin, protein engineering, phage display, staphylococcal display, inclusion bodies, refolding, proteomics, orthogonal affinity purification
National Category
Industrial Biotechnology Other Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-12823 (URN)978-91-7415-601-0 (ISBN)
Public defence
2010-05-28, D2, entréplan, Lindstedtsvägen 5, KTH, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

QC20100610

Available from: 2010-05-12 Created: 2010-05-12 Last updated: 2012-11-21Bibliographically 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. 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

Open Access in DiVA

No full text

Other links

Publisher's full textPubMedScopusBiotechnology Journal

Authority records BETA

Hedhammar, MyGräslund, TorbjörnHober, Sophia

Search in DiVA

By author/editor
Hedhammar, MyAlm, ToveGräslund, TorbjörnHober, Sophia
By organisation
Proteomics

Search outside of DiVA

GoogleGoogle Scholar

doi
pubmed
urn-nbn

Altmetric score

doi
pubmed
urn-nbn
Total: 120 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • harvard1
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf