Zhang, Yi2023-01-042023-01-042018-10https://hdl.handle.net/11299/250399University of Minnesota Ph.D. dissertation. 2018. Major: Chemistry. Advisor: Mark Distefano. 1 computer file (PDF); 168 pages.Over the years, protein-based conjugates have been extensively utilized in a variety of areas, including the study of fundamental biology as well as biotechnological and clinical applications. Compared to the non-selective conjugation methods which result in a mixture of modified products, site-specific modification by enzymatic methods provides homogeneously labeled proteins with identical physical properties and functional activities. In the first chapter of this dissertation, I reviewed the recent development (over the last five years) of enzymatic labeling techniques for a panel of ten different enzymes, including sortase A, subtiligase, microbial transglutaminase, protein farnesyltransferase (PFTase), N-myristoyltransferase, phosphopantetheinyl transferase, tubulin tyrosine ligase, lipoic acid ligase, biotin ligase and formylglycine generating enzyme. It is shown that each enzymatic method possesses its advantages and limitations and there is no single solution for all the applications. Therefore, both the protein substrates and the specific applications need to be considered to choose an optimal modification method. Among the ten enzymes being discussed above, PFTase is highly selective with fast kinetics. The enzyme transfers an isoprenoid moiety from farnesyl diphosphate to a C-terminal cysteine of the protein substrate. Consisted of only four amino acid residues, the enzyme recognition sequence is called CaaX-box, where C is the cysteine being modified. To label a protein of interest, a number of isoprenoid analogues bearing bioorthogonal functional groups have been developed, including azides, alkynes, and aldehydes. In this dissertation, I employed two different prenyltransferases to achieve dual- protein labeling and explored the applicability of using PFTase to modify several alternative binding proteins for the construction of targeted delivery systems, including the Designed Ankyrin Repeat Proteins (DARPins), the 10th extracellular domain of human fibronectin III (Fn3) and the nanobodies (VHH). It was found that all three binding proteins could be modified by PFTase successfully with high efficiency. Notably, the labeled DARPins retained the binding ability and specificity. To avoid complicated chromatographic purification process and streamline the production, a capture and release strategy was employed to create DARPin- fluorophore and DARPin-cytotoxin conjugates. Overall, this dissertation explored the utility of enzymatic labeling by PFTase towards various applications.enDARPinsenzymatic protein labelingfarnesyltransferasesite-specific protein modificationThesis or Dissertation