Protein Yield Testing for Adhesion Molecule Plasmids for Targeting Therapeutic Nanoparticles
Many studies have been done on different approaches for cancer therapy and one of the most promising fields is using nanoparticles as a drug delivery cancer therapeutic agent. The unique optical properties and high surface volume ratio of nanoparticles can improve the drug delivery system for not only cancer but other diseases. Moreover, nanoparticles can be attached to small proteins which provide the ability to target specific areas or inflamed tissues to minimize the death of healthy cells and tissues. Fusing the protein with fluorescent proteins also enables us to know the localization while targeting cells. Engineering a platform design for nanoparticles with adherent small proteins together with adding specific fluorescent proteins of interest on nanoparticles remains challenging. To achieve the goals, two different expression systems: E. coli and yeast protein expression system are being tested in this thesis. The E. coli expression system is known to have higher protein yield but with protein folding problems while the yeast expression system has lower yields but no protein folding problems which is easier for downstream applications. Therefore, the first part of this thesis is aiming to fuse different fluorescent proteins to an existing recombinant sequence which is construct in our lab and optimize the protein expression via E. coli and yeast expression system. The second part of the thesis is performing the protein yield testing of plasmids contains enzymatic tags. To perform the enzymatic test of our plasmid, we need to be sure that the enzymatic tags fused to our plasmid does not interact with the protein of interest which causes the protein yield to drop. Once we make sure that the enzymatic tags are not interfering the protein yield, enzymatic tests can be performed to know more about the enzyme activity our protein of interest and enzymes. Three different enzyme/tag systems are planned to be tested, Sfp synthase/S6 tag, and Sortase A/LPETG tag, and Lipoic Acid Ligase (LplA)/LAP2 tag. Different colors of fluorescent protein in this construct will create a library which enable us to have more options to choose while doing imaging and targeting in the future. It also creates a possibility to image multiple fluorescence simultaneously.