The structural core of the E2 subunit of B. stearothermophilus pyruvate dehydrogenase (E2) has been engineered to display antigens on its surface. For potential therapeutic applications, we have PEGylated a variant of E2 (E2-E279C) on introduced surface cysteines with methyl-PEG 24-maleimide. We verified PEGylation on the scaffold with mass spectrometry. Using dynamic light scattering and circular dichroism (CD), we showed the PEGylated proteins cage increased in size but the E2-E279C scaffold remained correctly folded. With CD thermostability test we confirmed that PEGylation dis not decrease the scaffold's high thermostability, with T m remaining near 90°C. Correct assembly was confirmed with transmission electron microscopy. We show that the designed E2-E279C mutant protein cage is chemically reactive toward thiol reactive compounds. Since application of E2-E279C protein cage enables easy and convenient “plug-and-play” surface modifications, we envision E2-E279C application not only as a drug delivery system, but also in imaging applications, cell targeting, scaffolds development for tissue engineering, and in vaccines design utilization.
|Commitee:||Da Silva, Nancy, Lee, Abraham|
|School:||University of California, Irvine|
|Department:||Biomedical Engineering - M.S.|
|School Location:||United States -- California|
|Source:||MAI 49/04M, Masters Abstracts International|
|Keywords:||Conjugation, Immunogenicity, Nanoparticle, Polyethylene glycol (PEG), Pyruvate dehydrogenase E2, Thiol|
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