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Development of a covalent site-specific antibody labeling strategy by the use of photoactivable Z domains
KTH, School of Biotechnology (BIO), Proteomics.
2012 (English)Licentiate thesis, comprehensive summary (Other academic)
Abstract [en]

The joining of two molecular functions or the strategy of adding functions to proteins has been tremendously important for the development of proteins as tools in research and clinic. Depending on the intended application, there are a wide variety of functions that can be added to a proteins. In clinical applications drugs are a commonly conjugated to antibodies and in research adding reporter groups such as biotin, enzymes or fluorophores is a routine procedure. The chemistries and methods most often used suffer from drawbacks such as lack of stringency, which could lead to undesired effects on the protein. Many site-specific methods of labeling of antibodies require modification or insertion of handles in the antibody recombinantly, before labeling can be performed.

The core of this thesis is the development of a strategy for covalent specific labeling of antibodies by exploiting the site specific binding of the Z domain to Protein A. Photoreactive Z-domains were produced by solid phase peptide synthesis, which provides the opportunity to insert a photoreactive amino acid and a reporter biotin at specific positions in the domain. The inherited binding to the Fc-part of the antibody in combination with the incorporated photoreactive amino acid, BPA, is used for site-specific interaction, and thereafter, covalent coupling to the antibody. The exposure with the appropriate wavelength of light enables the formation a covalent linkage between the Z domain and the antibody. The biotinylated photoactivable domains were subsequently used to site-specifically label a number of different types of antibodies, polyclonal rabbit IgG, monoclonal human IgG1 and monoclonal mouse IgG2a, and thereafter the antibodies was employed in a variation of applications. The photolabeling procedure of antibodies by the use of photoactivable Z domains has proven to be successful and could serve as a valuable tool in several applications.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2012. , 43 p.
Series
Trita-BIO-Report, ISSN 1654-2312 ; 2012:9
Keyword [en]
antibody labeling, site-specific, covalent, SPPS, Z domain, BPA
National Category
Engineering and Technology
Research subject
SRA - Molecular Bioscience
Identifiers
URN: urn:nbn:se:kth:diva-94007ISBN: 978-91-7501-329-9 (print)OAI: oai:DiVA.org:kth-94007DiVA: diva2:524969
Presentation
2012-05-25, FA32, Albanova Universitetscentrum, Roslagstullsbacken 21, Stockholm, 10:30 (English)
Opponent
Supervisors
Note
QC 20120507Available from: 2012-05-07 Created: 2012-05-04 Last updated: 2012-05-07Bibliographically approved
List of papers
1. Covalent Immunoglobulin Labeling through a Photoactivable Synthetic Z Domain
Open this publication in new window or tab >>Covalent Immunoglobulin Labeling through a Photoactivable Synthetic Z Domain
2011 (English)In: Bioconjugate chemistry, ISSN 1043-1802, E-ISSN 1520-4812, Vol. 22, no 12, 2395-2403 p.Article in journal (Refereed) Published
Abstract [en]

Traditionally, labeling of antibodies has been performed by covalent conjugation to amine or carboxyl groups. These methods are efficient but suffer from nonspecificity, since all free and available amine/carboxyl groups have the possibility to react. This drawback may lead to uncontrolled levels and locations of the labeling. Hence, the labeled molecules might behave differently and, possibly, the binding site of the antibody will also be affected. In this project, we have developed a highly stringent method for labeling of antibodies by utilizing an immunoglobulin-binding domain from protein A, the Z domain. Domain Z has been synthesized with an amino acid analogue, benzoylphenylalanine, capable of forming covalent attachment to other amino acids upon UV-exposure. This feature has been used for directed labeling of immunoglobulins and subsequent use of these in different assays.

National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-59002 (URN)10.1021/bc200052h (DOI)000298222300006 ()2-s2.0-84555195107 (Scopus ID)
Note
QC 20120110Available from: 2012-01-09 Created: 2012-01-09 Last updated: 2017-12-08Bibliographically approved
2. Antibodies biotinylated using a synthetic Z-domain provide stringent in situ proteindetection
Open this publication in new window or tab >>Antibodies biotinylated using a synthetic Z-domain provide stringent in situ proteindetection
Show others...
(English)Manuscript (preprint) (Other academic)
National Category
Biological Sciences
Identifiers
urn:nbn:se:kth:diva-94015 (URN)
Note
QS 2012Available from: 2012-05-04 Created: 2012-05-04 Last updated: 2012-05-07Bibliographically approved
3. Optimization of an antibody labeling strategy through the use of photoactivable synthetic Z domains
Open this publication in new window or tab >>Optimization of an antibody labeling strategy through the use of photoactivable synthetic Z domains
Show others...
(English)Manuscript (preprint) (Other academic)
National Category
Biological Sciences
Identifiers
urn:nbn:se:kth:diva-94030 (URN)
Note
QS 2012Available from: 2012-05-07 Created: 2012-05-07 Last updated: 2012-05-07Bibliographically approved

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