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
Staphylococcal surface display in directed evolution
KTH, School of Biotechnology (BIO), Molecular Biotechnology. (Molecular Biotechnology)
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Engineered affinity proteins have together with naturally derived antibodies becomeindispensable tools in many areas of life-science and with the increasing number ofapplications, the need for high-throughput methods for generation of such different affinityproteins is evident. Today, combinatorial protein engineering is the most successful strategy toisolate novel non-immunoglobulin affinity proteins. In this approach, generally termed directedevolution, high-complexity combinatorial libraries are created from which affinity proteins areisolated using an appropriate selection method, thus circumventing the need for detailedknowledge of the protein structure or the binding mechanism, often necessary in more rationalapproaches. Since the introduction of the phage display technology that pioneered the field ofcombinatorial engineering, several alternative selection systems have been developed for thispurpose.This thesis describes the development of a novel selection system based onstaphylococcal surface display and its implementation in directed evolution approaches. In thefirst study, the transformation efficiency to the gram-positive bacteria Staphylococcus carnosus wassuccessfully improved around 10,000-fold to a level that would allow cell surface display ofcomplex combinatorial protein libraries. In two separate studies, the staphylococcal displaysystem was investigated for the applicability in both de novo selection and affinity maturation ofaffibody molecules. First, using a pre-selection strategy with one round of phage display, ahigh-complexity affibody library was displayed on staphylococcal cells. Using fluorescenceactivatedcell sorting, binders with sub-nanomolar affinity to tumor necrosis factor-alpha(TNF-α) were isolated. Second, a combined approach using phage display for de novo selectionof first-generation affibody binders and staphylococcal display in a subsequent affinitymaturation selection was applied to generate binders with low nanomolar affinity to the humanepidermal growth factor receptor-3 (ErbB3). Moreover, in an additional study, thestaphylococcal surface display system was improved by the introduction of a protease 3Ccleavage sequence in the displayed fusion products in order to facilitate straightforwardproduction of soluble proteins for further downstream characterization.Altogether, the presented studies demonstrate that the staphylococcal selection systemindeed is a powerful tool for selection and characterization of novel affinity proteins and couldbecome an attractive alternative to existing selection techniques.

Place, publisher, year, edition, pages
Stockholm: KTH , 2009. , x, 78 p.
Series
Trita-BIO-Report, ISSN 1654-2312 ; 2009:16
Keyword [en]
affibody, combinatorial library, directed evoluation, Gram-positive bacteria. protein engineering
National Category
Industrial Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-11555ISBN: 978-91-7415-418-4 (print)OAI: oai:DiVA.org:kth-11555DiVA: diva2:277724
Public defence
2009-11-27, FD5, AlbaNova, Roslagstullsbacken 21, Stockholm, 10:15 (English)
Opponent
Supervisors
Note

QC 20100726

Available from: 2009-11-20 Created: 2009-11-20 Last updated: 2012-12-14Bibliographically approved
List of papers
1. Optimization of electroporation-mediated transformation: Staphylococcus carnosus as model organism
Open this publication in new window or tab >>Optimization of electroporation-mediated transformation: Staphylococcus carnosus as model organism
Show others...
2007 (English)In: Journal of Applied Microbiology, ISSN 1364-5072, E-ISSN 1365-2672, Vol. 102, no 3, 736-747 p.Article in journal (Refereed) Published
Abstract [en]

The study was conducted with an aim to optimize the transformation efficiency of the Gram-positive bacterium Staphylococcus carnosus to a level that would enable the creation of cell surface displayed combinatorial protein libraries.

Methods and Results: We have thoroughly investigated a number of different parameters for: (i) the preparation of electrocompetent cells; (ii) the treatment of cells before electroporation; (iii) the electroporation step itself; and (iv) improved recovery of transformed cells. Furthermore, a method for heat-induced inactivation of the host cell restriction system was devised to allow efficient transformation of the staphylococci with DNA prepared from other species, such as Escherichia coli. Previously described protocols for S. carnosus, giving transformation frequencies of approximately 10(2) transformants per transformation could be improved to reproducible procedures giving around 10(6) transformants for a single electroporation event, using plasmid DNA prepared from either S. carnosus or E. coli. The transformed staphylococcal cells were analysed using flow cytometry to verify that the entire cell population retained the introduced plasmid DNA and expressed the recombinant protein in a functional form on the cell surface at the same level as the positive control population.

Conclusions: The results demonstrate that the transformation frequency for S. carnosus could be dramatically increased through optimization of the entire electroporation process, and that the restriction barrier for interspecies DNA transfer, could be inactivated by heat treatment of the cells prior to electroporation.

Significance and Impact of the Study: The generation of large combinatorial protein libraries, displayed on the surface of S. carnosus can be envisioned in the near future, thus dramatically improving the selection compared with the traditional biopanning procedure used in phage display.

Keyword
affibody; cell surface display; electroporation; Gram positive; Staphylococcus carnosus; transformation
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-7845 (URN)10.1111/j.1365-2672.2006.03127.x (DOI)000244243900015 ()2-s2.0-33847018452 (Scopus ID)
Note
QC 20100726Available from: 2007-12-14 Created: 2007-12-14 Last updated: 2010-10-15Bibliographically approved
2. A novel affinity protein selection system based on staphylococcal cell surface display and flow cytometry
Open this publication in new window or tab >>A novel affinity protein selection system based on staphylococcal cell surface display and flow cytometry
Show others...
2008 (English)In: Protein Engineering Design & Selection, ISSN 1741-0126, E-ISSN 1741-0134, Vol. 21, no 4, 247-255 p.Article in journal (Refereed) Published
Abstract [en]

Here we describe the first reported use of a Gram-positive bacterial system for the selection of affinity proteins from large combinatorial libraries displayed on the surface of Staphylococcus carnosus. An affibody library of 3 x 109 variants, based on a 58 residue domain from staphylococcal protein A, was pre-enriched for binding to human tumor necrosis factor-alpha (TNF-alpha) using one cycle of phage display and thereafter transferred to the staphylococcal host (106 variants). The staphylococcal-displayed library was subjected to three rounds of flow-cytometric sorting, and the selected clones were screened and ranked by on-cell analysis for binding to TNF-alpha and further characterized using biosensor analysis and circular dichroism spectroscopy. The successful sorting yielded three different high-affinity binders (ranging from 95 pM to 2.2 nM) and constitutes the first selection of a novel affinity protein using Gram-positive bacterial display. The method combines the simplicity of working with a bacterial host with the advantages of displaying recombinant proteins on robust Gram-positive bacteria as well as using powerful flow cytometry in the selection and characterization process.

Keyword
affibody/cell surface display/combinatorial protein engineering/Gram-positive bacteria/Staphylococcus carnosus
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-7846 (URN)10.1093/protein/gzm090 (DOI)000254295200004 ()
Note
QC 20100722Available from: 2007-12-14 Created: 2007-12-14 Last updated: 2010-12-06Bibliographically approved
3. Simplified characterization through site-specific protease-mediated release of affinity proteins selected by staphylococcal display
Open this publication in new window or tab >>Simplified characterization through site-specific protease-mediated release of affinity proteins selected by staphylococcal display
Show others...
2008 (English)In: FEMS Microbiology Letters, ISSN 0378-1097, E-ISSN 1574-6968, Vol. 278, no 1, 128-136 p.Article in journal (Refereed) Published
Abstract [en]

The production of candidate affinity proteins in a soluble form, for downstream characterization, is often a time-consuming step in combinatorial protein engineering methods. Here, a novel approach for efficient production of candidate clones is described based on direct cleavage of the affinity protein from the surface of Staphylococcus carnosus, followed by affinity purification. To find a suitable strategy, three new fusion protein constructs were created, introducing a protease site for specific cleavage and purification tags for affinity chromatography purifications into the staphylococcal display vector. The three modified strains were evaluated in terms of transformation frequency, surface expression level and protease cleavage efficiency. A protocol for efficient affinity purification of protease-released affinity proteins using the introduced fusion-tags was successfully used, and the functionality of protease-treated and purified proteins was verified in a biosensor assay. To evaluate the devised method, a previously selected HER2-specific affibody was produced applying the new principle and was used to analyze HER2 expression on human breast cancer cells.

Keyword
Affibody, Combinatorial protein engineering, Gram-positive bacterial display, Protein production, Staphylococcus carnosus
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-7847 (URN)10.1111/j.1574-6968.2007.00990.x (DOI)000251386000019 ()18034830 (PubMedID)
Note
QC 20100726Available from: 2007-12-14 Created: 2007-12-14 Last updated: 2012-01-18Bibliographically approved
4. Combining phage and staphylococcal surface display for generation of ErbB3-specific Affibody molecules
Open this publication in new window or tab >>Combining phage and staphylococcal surface display for generation of ErbB3-specific Affibody molecules
Show others...
2011 (English)In: Protein Engineering Design & Selection, ISSN 1741-0126, E-ISSN 1741-0134, Vol. 24, no 4, 385-396 p.Article in journal (Refereed) Published
Abstract [en]

Emerging evidence suggests that the catalytically inactive ErbB3 (HER3) protein plays a fundamental role in normal tyrosine kinase receptor signaling as well as in aberrant functioning of these signaling pathways, resulting in several forms of human cancers. ErbB3 has recently also been implicated in resistance to ErbB2-targeting therapies. Here we report the generation of high-affinity ErbB3-specific Affibody molecules intended for future molecular imaging and biotherapeutic applications. Using a high-complexity phage-displayed Affibody library, a number of ErbB3 binders were isolated and specific cell-binding activity was demonstrated in immunofluorescence microscopic studies. Subsequently, a second-generation library was constructed based on sequences of the candidates from the phage display selection. By exploiting the sensitive affinity discrimination capacity of a novel bacterial surface display technology, the affinity of candidate Affibody molecules was further increased down to subnanomolar affinity. In summary, the demonstrated specific targeting of native ErbB3 receptor on human cancer cell lines as well as competition with the heregulin/ErbB3 interaction indicates that these novel biological agents may become useful tools for diagnostic and therapeutic targeting of ErbB3-expressing cancers. Our studies also highlight the powerful approach of combining the advantages of different display technologies for generation of functional high-affinity protein-based binders. Potential future applications, such as radionuclide-based diagnosis and treatment of human cancers are discussed.

Keyword
Affibody, cell surface display, combinatorial protein engineering, HER3, Staphylococcus carnosus
National Category
Condensed Matter Physics
Identifiers
urn:nbn:se:kth:diva-14208 (URN)10.1093/protein/gzq118 (DOI)000288269600005 ()
Funder
Swedish Research Council, 2009-5758
Note

QC 20100726 Uppdaterad från manuskript till artikel i tidskrift (20110408)

Available from: 2010-07-26 Created: 2010-07-26 Last updated: 2017-12-12Bibliographically approved

Open Access in DiVA

fulltext(9119 kB)663 downloads
File information
File name FULLTEXT01.pdfFile size 9119 kBChecksum SHA-512
1d3a5e94ac9bb1b2e04482bf1341284510a25fcee0d39e7b652fe0f32b17392a3d90f823aaed863c3c0e75f5b25eb9aeccf678ef8d1541d7e93bbe82f181772c
Type fulltextMimetype application/pdf

Search in DiVA

By author/editor
Kronqvist, Nina
By organisation
Molecular Biotechnology
Industrial Biotechnology

Search outside of DiVA

GoogleGoogle Scholar
Total: 663 downloads
The number of downloads is the sum of all downloads of full texts. It may include eg previous versions that are now no longer available

isbn
urn-nbn

Altmetric score

isbn
urn-nbn
Total: 341 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