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Chemical Synthesis of Triple-Labelled Three-Helix Bundle Binding Proteins for Specific Fluorescent Detection of Unlabelled Protein
KTH, School of Biotechnology (BIO).
KTH, School of Biotechnology (BIO).
KTH, School of Biotechnology (BIO).
KTH, School of Biotechnology (BIO).ORCID iD: 0000-0003-4214-6991
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2005 (English)In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 6, no 6, 1043-1050 p.Article in journal (Refereed) Published
Abstract [en]

Site-specifically triple-labelled three-helix bundle affinity proteins (affibody molecules) have been produced by total chemical Synthesis. The 58 aa affinity proteins were assembled on an automated peptide synthesizer, followed by manual on-resin incorporation of three different reporter groups. An orthogonal protection strategy was developed for the site-specific introduction of 5-(2-aminethylamino)-1-nophthalenesulfonic acid (EDANS) and 6(7-nitrobenzofurazon-4-yiamino)-hexanoic acid (NBDX), constituting a donor/acceptor pair for fluorescence resonance energy transfer (FRET), and a biotin moiety, used for surface immobilization. Circular dichroism and biosensor studies of the synthetic proteins and their recombinant counterparts revealed that the synthetic proteins were folded and retained their binding specificities. The biotin-conjugated protein could be immobilized onto a streptavidin surface without loss of activity. The synthetic, doubly fluorescent-labelled affinity proteins were shown to function as fluorescent biosensors in an assay for the specific detection of unlabelled human IgG and IgA.

Place, publisher, year, edition, pages
2005. Vol. 6, no 6, 1043-1050 p.
Keyword [en]
affinity proteins; biosensors; FRET; peptides; protein modifications; solid-phase synthesis
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:kth:diva-5646DOI: 10.1002/cbic.200400388ISI: 000229730000015Scopus ID: 2-s2.0-20444507608OAI: oai:DiVA.org:kth-5646DiVA: diva2:10083
Note
QC 20100715Available from: 2008-12-03 Created: 2008-12-03 Last updated: 2010-09-16Bibliographically approved
In thesis
1. Chemical Synthesis of Affibody Molecules for Protein Detection and Molecular Imaging
Open this publication in new window or tab >>Chemical Synthesis of Affibody Molecules for Protein Detection and Molecular Imaging
2008 (English)Doctoral thesis, comprehensive summary (Other scientific)
Abstract [en]

Proteins are essential components in most processes in living organisms. The detection and quantification of specific proteins can be used e.g. as measures of certain physiological conditions, and are therefore of great importance. This thesis focuses on development of affinity-based bioassays for specific protein detection. The use of Affibody molecules for specific molecular recognition has been central in all studies in this thesis. Affibody molecules are affinity proteins developed by combinatorial protein engineering of the 58-residue protein A-derived Z domain scaffold. In the first paper, solid phase peptide synthesis is investigated as a method to generate functional Affibody molecules. Based on the results from this paper, chemical synthesis has been used throughout the following papers to produce Affibody molecules tailored with functional groups for protein detection applications in vitro and in vivo.

 

In paper I, an orthogonal protection scheme was developed to enable site-specific chemical introduction of three different functional probes into synthetic Affibody molecules. Two of the probes were fluorophores that were used in a FRET-based binding assay to detect unlabeled target proteins. The third probe was biotin, which was used as an affinity handle for immobilization onto a solid support. In paper II, a panel of Affibody molecules carrying different affinity handles were synthesized and evaluated as capture ligands on microarrays. Paper III describes the synthesis of an Affibody molecule that binds to the human epidermal growth factor receptor type 2, (HER2), and the site-specific incorporation of a mercaptoacetyl-glycylglycylglycine (MAG3) chelating site in the peptide sequence to allow for radiolabeling with 99mTc. The derivatized Affibody molecule was found to retain its binding capacity, and the 99mTc-labeling was efficient and resulted in a stable chelate formation. 99mTc-labeled Affibody molecules were evaluated as in vivo HER2-targeting imaging agents in mice. In the following studies, reported in papers IV-VI, the 99mTc-chelating sequence was engineered in order to optimize the pharmacokinetic properties of the radiolabeled Affibody molecules and allow for high-contrast imaging of HER2-expressing tumors and metastatic lesions. The main conclusion from these investigations is that the biodistribution of Affibody molecules can be dramatically modified by amino acid substitutions directed to residues in the MAG3-chelator. Finally, paper VII is a report on the chemical synthesis and chemoselective ligation to generate a cross-linked HER2-binding Affibody molecule with improved thermal stability and tumor targeting capacity.

 

Taken together, the studies presented in this thesis illustrate how peptide synthesis can be used for production and modification of small affinity proteins, such as Affibody molecules for protein detection applications.

Place, publisher, year, edition, pages
Stockholm: KTH, 2008. viii, 84 p.
Series
Trita-BIO-Report, ISSN 1654-2312 ; 2008:22
Keyword
Affibody, peptide synthesis, protein detection, molecular imaging
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-9626 (URN)978-91-7415-152-7 (ISBN)
Public defence
2008-12-12, D2, Lindstedtsv 5, Stockholm, 10:00 (English)
Opponent
Supervisors
Note
QC 20100719Available from: 2008-11-21 Created: 2008-11-21 Last updated: 2010-07-19Bibliographically approved
2. Fluorescence-based ligand assays for protein detection using affibody affinity proteins
Open this publication in new window or tab >>Fluorescence-based ligand assays for protein detection using affibody affinity proteins
2006 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The detection and quantification of biomolecules, and proteins in particular, are of great interest since these molecules are of fundamental importance to our well-being. Body fluids, as for instance human blood, are well suited for sampling of protein levels. However, the complexity of the fluids and the low abundance of many of the interesting biomolecules makes detection and quantification difficult. This has spurred an interest into the development of many protein detection methods, and of these, ligand assays have proven particularly suitable. In this thesis, different types of ligand assays for protein detection have been developed using affibody molecules as ligands.

In a first study, a homogeneous competitive detection assay was investigated, based on antiidiotypic affibody molecule pairs and fluorescence resonance energy transfer (FRET) as reporting system. The individual members of two anti-idiotypic affibody pairs, each consisting of a target binding (idiotypic) and an anti-idiotypic affibody ligand, were labeled with a donor fluorophore and an acceptor fluorophore, respectively. Incubation with the two target proteins IgA and Taq DNA polymerase resulted in a concentration dependent decrease in the FRET signal, allowing for target protein detection and quantification. For Taq DNA polymerase, detection in 25% human plasma was also possible in the same concentration span as in buffer.

In a second study, a homogeneous, non-competitive detection system was described. Affibody molecules of 58 amino acids directed against IgA and IgG were produced with chemical synthesis, and two fluorophores capable of FRET were site-specifically introduced. Binding of target protein induced a concentration-dependent change in the relative emission of the two fluorophores, which formed the basis for the detection system.

In two studies, affibody molecules were evaluated and shown to function well as capture ligands on microarrays. Synthetic affibody molecules directed against Taq DNA polymerase and IgA were modified by the introduction of immobilization tags. Specific immobilization via a C-terminal cysteine or a biotin moiety, or random immobilization via amino groups, were studied in protein microarray experiments and SPR-based biosensor studies. The experiments showed that all immobilization chemistries resulted in functional capture molecules. A short spacer was also introduced, situated between the affibody and the cysteine and biotin moieties, which was shown to improve binding for all constructs. Multidomain affibody constructs of up to four N- to C-terminally linked domains were shown to increase the amount of bound target, compared to monomeric affibody ligands. Six dimeric affibody constructs directed against IgA, IgG, IgE, Taq DNA polymerase, TNF-α and insulin, respectively, showed low limits of detections for their targets and little or no cross-reactivity with the other target proteins. Dimeric affibody molecules directed against IgA and TNF-α were also shown to function in a sandwich format with antibodies for detection of targets in buffer and in human serum and plasma. Successful discrimination between normal and IgA-deficient sera showed that affibody molecules could be used for specific detection of protein in highly complex backgrounds on microarrays.

Place, publisher, year, edition, pages
Stockholm: KTH, 2006. 94 p.
Keyword
Affibody molecules, biosensors, FRET, immobilisation, solid-phase synthesis, protein microarrays
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-3936 (URN)91-7178-344-X (ISBN)
Public defence
2006-05-19, FR4, Albanova Universitetscentrum, Roslagstullsbacken 21, Stockholm, 13:00
Opponent
Supervisors
Note
QC 20100916Available from: 2006-05-05 Created: 2006-05-05 Last updated: 2011-12-08Bibliographically approved

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Nygren, Per-ÅkeEriksson Karlström, Amelie

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