Generation and characterization of a prostate-specific membrane antigen positive eukaryotic cell system for phage selection

Sammanfattning: Prostate cancer is one of the most common cancer types worldwide. However, current diagnostic approaches and treatments are invasive and unspecific. Prostate-specific membrane antigen (PSMA) is an ideal biomarker for prostate cancer and can act as a target for therapeutic or diagnostic agents. Previous attempts to develop an affibody with affinity towards PSMA have been unsuccessful, therefore this thesis aimed at making the affibody selections against PSMA more efficient. In this thesis HEK293 cells expressing a modified version of PSMA containing a 3C protease cleavage site were generated, to enable extraction of the extracellular domain of PSMA during the selections. However, further analyses must be performed to determine if the extracellular domain can be successfully cleaved off. To develop an affibody that can be used both in vitro and in vivo, selections will be carried out against recombinant PSMA as well. The recombinant PSMA was previously produced incorporating an Avi tag for site-specific biotinylation and immobilization for the selections. To biotinylate the recombinant PSMA, the enzyme BirA that catalyzes the biotinylation of the Avi tag, was produced. A protein yield of 8.95 mg/liter culture was obtained and the site-specific biotinylation was highly efficient. To evaluate the proposed affibody selection strategy the next step is to determine if cleavage of the PSMA expressed on the HEK293 cells is possible, optimize the cleavage conditions and to start initial selections using the generated HEK293 cells and the produced BirA enzyme.