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A novel affinity protein selection system based on staphylococcal cell surface display and flow cytometry
KTH, School of Biotechnology (BIO), Molecular Biotechnology.
KTH, School of Biotechnology (BIO), Molecular Biotechnology.ORCID iD: 0000-0001-9423-0541
KTH, School of Biotechnology (BIO), Molecular Biotechnology.
KTH, School of Biotechnology (BIO), Molecular Biotechnology.ORCID iD: 0000-0002-9282-0174
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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.

Place, publisher, year, edition, pages
2008. Vol. 21, no 4, 247-255 p.
Keyword [en]
affibody/cell surface display/combinatorial protein engineering/Gram-positive bacteria/Staphylococcus carnosus
National Category
Industrial Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-7846DOI: 10.1093/protein/gzm090ISI: 000254295200004OAI: oai:DiVA.org:kth-7846DiVA: diva2:12987
Note
QC 20100722Available from: 2007-12-14 Created: 2007-12-14 Last updated: 2010-12-06Bibliographically approved
In thesis
1. Development of molecular recognition by rational and combinatorial engineering
Open this publication in new window or tab >>Development of molecular recognition by rational and combinatorial engineering
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Combinatorial protein engineering, taking advantage of large libraries of protein variants and powerful selection technology, is a useful strategy for developing affinity proteins for applications in biotechnology and medicine. In this thesis, two small affinity proteins have been subjected to combinatorial protein engineering to improve or redirect the binding. In two of the projects, a three-helix protein domain based on staphylococcal protein A has been used as scaffold to generate so called Affibody molecules capable of binding to key proteins related to two diseases common among elderly people.

In the first project, Affibody molecules were selected using phage display technology for binding to Ab-peptides, believed to play a crucial role in Alzheimer’s disease, in that they can oligomerize and contribute to the formation of neural plaques in the brain. The selected Affibody molecules were found to efficiently capture Ab from spiked human plasma when coupled to an affinity resin. The structure of the complex was determined by nuclear magnetic resonance (NMR) and demonstrated that the original helix 1 in the two Affibody molecules was unfolded upon binding, forming intermolecular b-sheets that stabilized the Ab peptide as buried in a tunnel-like cavity. Interestingly, the complex structure also revealed that the Affibody molecules were found to homo-dimerize via a disulfide bridge and bind monomeric Ab-peptide with a 2:1 stoichiometry. Furthermore, Affibody molecule-mediated inhibition of Ab fibrillation in vitro, suggested a potential of selected binders for future therapeutic applications.

In the second project, two different selection systems were used to isolate Affibody molecules binding to tumor necrosis factor alpha (TNF), which is involved in inflammatory diseases such as rheumatoid arthritis. Both selection systems, phage display and Gram-positive bacterial display, could successfully generate TNF-binding molecules, with equilibrium dissociation constants (KD) in the picomolar to nanomolar range. Initial characterization of the binding to TNF was evaluated by competitive binding studies between the Affibody molecules and clinically approved TNF antagonists (adaliumumab, infliximab and etanercept) and demonstrated overlapping binding sites with both adaliumumab and etanercept. Furthermore, linkers of different lengths were introduced between Affibody moieties, in dimeric and trimeric constructs that were evaluated for their ability to block the binding between TNF and a recombinant form of its receptor. In the dimeric constructs, a linker length of 20-40 amino acids seemed to have an advantage compared to shorter and longer linkers, and the tested trimeric construct could block the TNF binding at even lower concentration. The results provided valuable information for the design of future Affibody-based molecules that could be investigated in therapeutic or medical imaging applications.

In the third project aiming to generate a protein domain with capacity to influence the pharmacokinetics of protein therapeutics, a natural serum albumin-binding domain (ABD) was subjected to an engineering effort aiming at improving the affinity to human serum albumin (HSA), a protein with an exceptional long half-life in serum (19 days). First-generation affinity improved ABD variants were selected using phage display technology from a constructed ABD library. After additional rational engineering of such first generation variants, one variant with a 10,000-fold improved affinity to HSA (KD ≈ 120 fM) was obtained. Furthermore, characterization of this molecule also demonstrated improved affinity to several other serum albumins. When used as a gene fusion partner, this affinity-maturated variant denoted ABD035, should have the potential to extend the half-life of biopharmaceuticals in humans, and several other animal species.

Place, publisher, year, edition, pages
Stockholm: KTH, 2009. 94 p.
Series
TRITA BIO-Report 2009:2, ISSN 1654-2312 ; 2009 : 2
Keyword
Affibody molecule, albumin binding domain, protein engineering, phage display, amyloid beta peptide, TNF, HSA
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-10038 (URN)978-91-7415-238-8 (ISBN)
Public defence
2009-03-27, sal E3, Osquarsbacke 14, Stockholm, 10:00 (Swedish)
Opponent
Supervisors
Note
QC 20100722Available from: 2009-03-10 Created: 2009-03-05 Last updated: 2011-11-24Bibliographically approved
2. Staphylococcal surface display in directed evolution
Open this publication in new window or tab >>Staphylococcal surface display in directed evolution
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
affibody, combinatorial library, directed evoluation, Gram-positive bacteria. protein engineering
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-11555 (URN)978-91-7415-418-4 (ISBN)
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
3. 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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Publisher's full texthttp://peds.oxfordjournals.org/cgi/content/abstract/gzm090v1

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Löfblom, JohnStåhl, Stefan

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