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Fine affinity discrimination by normalized fluorescence activated cell sorting in staphylococcal surface display
KTH, School of Biotechnology (BIO).ORCID iD: 0000-0001-9423-0541
KTH, School of Biotechnology (BIO).
KTH, School of Biotechnology (BIO).ORCID iD: 0000-0002-9282-0174
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.

Place, publisher, year, edition, pages
2005. Vol. 248, no 2, 189-198 p.
Keyword [en]
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: urn:nbn:se:kth:diva-7843DOI: 10.1016/j.femsle.2005.05.040ISI: 000230635200009PubMedID: 15964717Scopus ID: 2-s2.0-21744460508OAI: oai:DiVA.org:kth-7843DiVA: diva2:12984
Note
QC 20100809Available from: 2007-12-14 Created: 2007-12-14 Last updated: 2017-12-14Bibliographically approved
In thesis
1. Staphylococcal surface display for protein engineering and characterization
Open this publication in new window or tab >>Staphylococcal surface display for protein engineering and characterization
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
affibody, combinatorial library, epitope mapping, Gram-positive bacteria, protein engineering, staphylococcal surface display
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-4584 (URN)978-91-7178-834-4 (ISBN)
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

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Löfblom, JohnStåhl, Stefan

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