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Staphylococcal surface display for protein engineering and characterization
KTH, School of Biotechnology (BIO).ORCID iD: 0000-0001-9423-0541
2007 (English)Doctoral thesis, comprehensive summary (Other scientific)
Abstract [en]

Even though our understanding of mechanisms such as protein folding and molecular recognition is relatively poor, antibodies and alternative affinity proteins with entirely novel functions are today generated in a routine manner. The reason for this success is an engineering approach generally known as directed evolution.

Directed evolution has provided researchers with a tool for circumventing our limited knowledge and hence the possibility to create novel molecules that by no means could have been designed today. The approach is based on construction of high-complexity combinatorial libraries from which protein variants with desired properties can be selected. Engineered proteins are already indispensable tools in nearly all areas of life science and the recent advent of mainly monoclonal antibodies as therapeutic agents has directed even more attention to the field of combinatorial protein engineering.

In this thesis, I present the underlying research efforts of six original papers. The overall objective of the studies has been to develop and investigate a new staphylococcal surface display method for protein engineering and protein characterization. The technology is based on display of recombinant proteins on surface of the Gram-positive bacteria Staphylococcus carnosus. In two initial studies, two key issues were addressed in order to improve the protein engineering method in regard to affinity discrimination ability and transformation efficiency. The successful results enabled investigation of the staphylococcal display system for de novo generation of affibody molecules from large combinatorial libraries. In this study, a high-complexity protein library was for the first time displayed on surface of Gram-positive bacteria and by means of fluorescence-activated cell sorting, specific affinity proteins for tumor necrosis factor-alpha were isolated. Moreover, in following papers, the staphylococcal display method was further improved and investigated for affinity determination, soluble protein production and epitope mapping purposes in order to facilitate downstream characterizations of generated affinity proteins.

Taken together, in these studies we have demonstrated that the staphylococcal display system is a powerful alternative to existing technologies for protein engineering and protein characterization.

Place, publisher, year, edition, pages
Stockholm: KHT , 2007. , x, 95 p.
Keyword [en]
affibody, combinatorial library, epitope mapping, Gram-positive bacteria, protein engineering, staphylococcal surface display
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:kth:diva-4584ISBN: 978-91-7178-834-4 (print)OAI: oai:DiVA.org:kth-4584DiVA: diva2:12990
Public defence
2008-01-11, FD5, Albanova, Roslagstullsbacken 21, Stockholm, 09:00
Opponent
Supervisors
Note
QC 20100809Available from: 2007-12-14 Created: 2007-12-14 Last updated: 2010-08-09Bibliographically approved
List of papers
1. Fine affinity discrimination by normalized fluorescence activated cell sorting in staphylococcal surface display
Open this publication in new window or tab >>Fine affinity discrimination by normalized fluorescence activated cell sorting in staphylococcal surface display
2005 (English)In: FEMS Microbiology Letters, ISSN 0378-1097, E-ISSN 1574-6968, Vol. 248, no 2, 189-198 p.Article in journal (Refereed) Published
Abstract [en]

We have investigated a staphylococcal surface display system for its potential future use as a protein library display system ill combinatorial biochemistry. Efficient affinity-based selections require a system capable of fine affinity discrimination of closely related binders to minimize the loss of potentially improved variants. In this Study, a significant breakthrough was achieved to avoid biases due to potential cell-to-cell variations in surface expression levels, since it was found that a generic protein tag, present within the displayed recombinant surface proteins on the cells, could be successfully employed to obtain normalization of the target-binding signal. Four mutated variants of a staphylococcal protein A domain with different affinity to human IgG were successfully expressed on the surface of recombinant Staphylococcus carnosus cells. The system was evaluated for affinity-based cell sorting experiments, where cell-displayed protein A domains with an 8-fold difference in target affinity were mixed at a ratio of 1: 1000 and sorted using FACS. Enrichment factors around 140-fold were obtained from a single round of sorting under normal library sorting conditions when the top 0.1% fraction having the highest antigen binding to Surface expression level ratio was sorted. The results demonstrate that the system would have a potential as a selection system in protein library display applications, and the normalization strategy should indeed make it possible to achieve fine affinity discriminations in future library selections. (c) 2005 Federation of European Microbiological Societies.

Keyword
Affibody; Affinity discrimination; Cell surface display; FACS; Gram positive; Staphylococcus carnosus; immunoglobulin G; membrane protein; antibody combining site; article; binding affinity; cell interaction; cellular distribution; evaluation; fluorescence activated cell sorting; nonhuman; priority journal; protein domain; protein expression; protein localization; protein targeting; signal transduction; Staphylococcus; Staphylococcus carnosus; surface property; Amino Acid Sequence; Antibody Affinity; Flow Cytometry; Immunoglobulin G; Molecular Sequence Data; Mutation; Peptide Library; Protein Structure, Tertiary; Recombinant Proteins; Sequence Alignment; Staphylococcal Protein A; Staphylococcus; Transformation, Bacterial; Posibacteria; Staphylococcus carnosus
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-7843 (URN)10.1016/j.femsle.2005.05.040 (DOI)000230635200009 ()15964717 (PubMedID)2-s2.0-21744460508 (Scopus ID)
Note
QC 20100809Available from: 2007-12-14 Created: 2007-12-14 Last updated: 2010-08-09Bibliographically approved
2. Evaluation of staphylococcal cell surface display and flow cytometry for postselectional characterization of affinity proteins in combinatorial protein engineering applications
Open this publication in new window or tab >>Evaluation of staphylococcal cell surface display and flow cytometry for postselectional characterization of affinity proteins in combinatorial protein engineering applications
2007 (English)In: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, Vol. 73, no 21, 6714-6721 p.Article in journal (Refereed) Published
Abstract [en]

For efficient generation of high-affinity protein-based binding molecules, fast and reliable downstream characterization platforms are needed. In this work, we have explored the use of staphylococcal cell surface display together with How cytometry for affinity characterization of candidate affibody molecules directly on the cell surface. A model system comprising three closely related affibody molecules with different affinities for immunoglobulin G and an albumin binding domain with affinity for human serum albumin was used to investigate advantages and differences compared to biosensor technology in a side-by-side manner. Equilibrium dissociation constant (K-D) determinations as well as dissociation rate analysis were performed using both methods, and the results show that the on-cell determinations give both KD and dissociation rate values in a very fast and reproducible manner and that the relative affinities are very similar to the biosensor results. Interestingly, the results also show that there are differences between the absolute affinities determined with the two different technologies, and possible explanations for this are discussed. This work demonstrates the advantages of cell surface display for directed evolution of affinity proteins in terms of fast postselectional, on-cell characterization of candidate clones without the need for subcloning and subsequent protein expression and purification but also demonstrates that it is important to be aware that absolute affinities determined using different methods often vary substantially and that such comparisons therefore could be difficult.

Keyword
Biosensors; Characterization; Cytology; Flow cytometry; Gene expression; Proteins; Reaction rates; Affibody molecules; Binding molecules; Cell surface; Immunoglobulins; Staphylococcal cell surface; Biochemical engineering; albumin; immunoglobulin G; bacterium; cell organelle; flow cytometry; protein; article; binding affinity; cell surface; combinatorial chemistry; display system; dissociation constant; equilibrium constant; flow cytometry; molecular cloning; molecular model; nonhuman; protein binding; protein domain; protein engineering; protein expression; protein purification; reliability; reproducibility; Staphylococcus; surface property; Antibodies; Bacterial Outer Membrane Proteins; Binding Sites; Biosensing Techniques; Flow Cytometry; Humans; Immunoglobulin G; Protein Engineering; Staphylococcus
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-7844 (URN)10.1128/AEM.01432-07 (DOI)000250700600003 ()17873070 (PubMedID)2-s2.0-35948962788 (Scopus ID)
Note
QC 20100809Available from: 2007-12-14 Created: 2007-12-14 Last updated: 2010-08-09Bibliographically approved
3. 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
4. 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
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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
5. 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
6. Epitope mapping of antibodies using bacterial surface display
Open this publication in new window or tab >>Epitope mapping of antibodies using bacterial surface display
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2008 (English)In: Nature Methods, ISSN 1548-7091, E-ISSN 1548-7105, Vol. 5, no 12, 1039-1045 p.Article in journal (Refereed) Published
Abstract [en]

We describe a method for mapping the epitopes recognized by antibodies, based on bacterial surface expression of antigen protein fragments followed by antibody-based flow-cytometric sorting. We analyzed the binding sites of both monoclonal and polyclonal antibodies directed to three human protein targets: (i) the human epidermal growth factor receptor 2 (HER2), (ii) ephrin-B3 and (iii) the transcription factor SATB2. All monoclonal antibodies bound a single epitope, whereas the polyclonal antibodies showed, in each case, a binding pattern with one to five separate epitopes. A comparison of polyclonal and monoclonal antibodies raised to the same antigen showed overlapping binding epitopes. We also demonstrated that bacterial cells with displayed protein fragments can be used as affinity ligands to generate epitope-specific antibodies. Our approach shows a path forward for systematic validation of antibodies for epitope specificity and cross-reactivity on a whole-proteome level.

Keyword
ephrin B3; epidermal growth factor receptor 2; epitope; monoclonal antibody; polyclonal antibody; proteome; transcription factor; transcription factor SATB2; unclassified drug; antibody affinity; antibody labeling; antibody specificity; antigen antibody complex; article; bacterial cell; binding affinity; binding site; controlled study; cross reaction; epitope mapping; flow cytometry; host cell; ligand binding; nonhuman; priority journal; protein targeting; Staphylococcus carnosus; Antibodies; Biological Assay; Cell Membrane; Epitope Mapping; Protein Engineering; Staphylococcus; Bacteria (microorganisms)
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-7848 (URN)10.1038/nmeth.1272 (DOI)000261212700018 ()19029907 (PubMedID)
Note
QC 20100809. Uppdaterad från Submitted till Published. Titel ändrad, tidigare titel: "Combinatorial epitope mapping of antibodies using staphylococcal surface display" 20100809.Available from: 2007-12-14 Created: 2007-12-14 Last updated: 2012-01-11Bibliographically approved

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