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Development of a Site-Specific Albumin-Binding Assay

Research output: Book/anthology/dissertation/reportPh.D. thesisResearch

Human Serum Albumin is a ~66 kDa protein with an average concentration of 0.6 mM in the blood circulation system. The high abundance is mainly due to its long circulatory half-life of approximately 19 days and is connected with its function as a carrier and distributer of endogenous ligands such as fatty acids, other proteins and small molecules. The numerous binding sites of albumin are well distributed as pockets in the structure of albumin and due to high plasticity, the pockets can accommodate a variety of different ligands. The favourable physiological properties of albumin make it a highly attractive target for circulatory half-life extension of biopharmaceuticals. To understand the pharmacokinetic properties of drug delivery, a pocket-specific characterisation of binding modes is highly desirable and has to date not been reported.
In this project, we aimed to develop a Site-Specific Albumin Binding Assay using liquid state 2D Nuclear Magnetic Resonance Spectroscopy (NMR). The assay required recombinant production of residue specific isotope-labelled albumin mutants with a deuterated background. This was sought with E. coli and finally attained with P. pastoris as expression hosts. NMR signals of selected methyl-groups would pose as probes and chemical shift perturbations would report of a change in their local chemical and magnetic environment. The signals were mapped to the protein structure through assignment by mutagenesis, which was achieved for all methionine residues of albumin that at this point constitute the Site-Specific Albumin Binding Assay. The assay was verified by titration experiments with multiple known ligands, including small-molecule drugs and fatty acids. Since the results are very promising, the assay ought to be expanded with more assigned methyl-groups and complimented by other methods.
Original languageEnglish
PublisherAarhus Universitet
Number of pages148
Publication statusPublished - 2020

Note re. dissertation

FRE 29 MAJ 2020<br/>13:00-15:00 | Afholdes online<br/>Ph.d.-forsvar: Malene Selch Sønnichsen (Vejleder: Frans Mulder)<br/>Udvikling af et Site-Specifikt Albumin-bindings Assay<br/>Termination date: 17.06.2020

    Research areas

  • albumin, NMR, site specific

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