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. / QC 20120507
| Identifer | oai:union.ndltd.org:UPSALLA1/oai:DiVA.org:kth-94007 |
| Date | January 2012 |
| Creators | Konrad, Anna |
| Publisher | KTH, Proteomik, Stockholm |
| Source Sets | DiVA Archive at Upsalla University |
| Language | English |
| Detected Language | English |
| Type | Licentiate thesis, comprehensive summary, info:eu-repo/semantics/masterThesis, text |
| Format | application/pdf |
| Rights | info:eu-repo/semantics/openAccess |
| Relation | Trita-BIO-Report, 1654-2312 ; 2012:9 |
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