|
|
Modulation of interactions between interleukins and their receptors
Nepokojová, Tereza ; Schneider, Bohdan (advisor) ; Obšilová, Veronika (referee)
Scaffolds are proteins with high conformational stability, allowing us to implement multiple mutations into specific parts of the protein. Even with these mutations, the structural integrity of the protein is maintained as well as its physical-chemical properties. These mutations give the specific scaffold new properties. In most cases it is the binding specificity towards previously chosen target. The biggest advantages of scaffolds are their small size, stability, low-cost manufacturing, and easiness of preparation. Scaffold utilized in this thesis is unique for having two binging surfaces designed on which it can be mutated. Each of those two surfaces can be separately mutated to develop a binging site for two different proteins. In our case these mutations led to binding two nonidentical receptors of a human cytokine. Mutations are made with a use of yeast display, one of the methods of directed evolution. The main focus of this thesis is changing an expression system of the binding proteins from the yeast system to a bacterial one, their production and purification followed by characterization of those binding proteins using biophysical methods. These methods were used to evaluate structural and thermal stability, and binding affinity to both receptors of the beforementioned binding proteins....
|
|
|
Selection approaches in directed evolution of binding proteins
Huličiak, Maroš ; Malý, Petr (advisor) ; Hlouchová, Klára (referee)
Artificial binding proteins derived from small protein domains attract attention as a promising alternative to monoclonal antibodies and can be used in many kinds of applications. They are useful in diagnosis of human diseases, seem to be a clue for more efficient vaccine development preventing from global diseases such as AIDS, can exhibit a therapeutic potential or improve purification techniques. For the selection of protein variants with desired properties such as high specificity and binding affinity, more than 10 different selection techniques have been developed. So called display techniques such as phage display, yeast display, retroviral display or baculovirus display are based on protein expression from different vectors. Contrary that, ribosome display, mRNA display and CIS display are cell-free systems based on in vitro translation. Development of different selection approaches allows production of post- translationally glycosylated, phosphorylated and acetylated proteins, increased yield of the produced binders and improved their binding properties. The submitted work provides an overview of current selection techniques, compare their parametres regarding to combinatorial libraries, describes their advantageous properties and limitations, and focus on a practical utilization of...
|
guest ::
