Abstract:
Cancer is a vast group of diseases caused by mutations in the human DNA that result in an uncontrollable growth of cells which can spread around the body. Cancer is one of the leading causes of mortality worldwide.
Advancements in CTXs allowed the development of effective treatments. However, most CTX agents present poor pharmacokinetic and safety profiles. Development of appropriate drug carriers is one of the potential solutions for counteracting these drawbacks.
The aim of this thesis is to make advancements in the engineering of a protein based carrier, with higher binding affinity to the CTX agents. Furthermore, explore the expression of fusion proteins with the aim of improving the characteristics of the delivery system.
In the first phase of the project, 55 genes, corresponding to human serum albumin (HSA) mutants previously engineered to have higher binding affinity to doxorubicin or 9-aminocamptothecin, were cloned by Gibson assembly (GA). 52 of these mutants were then successfully expressed in a yeast display system, which will be used for future binding affinity experiments.
For the second part of this thesis, a cloning system using restriction enzymes (RE) was designed in order to produce a series of HSA oligomers. The HSA dimer was successfully cloned, expressed, and purified. However it showed a complete degradation over time. Therefore, alternative cloning strategies or changes in the design of the linker between the monomers should be employed.
