| This thesis describes the development and application of chemical complementation, a high-throughput assay that can be used for enzyme evolution and proteomics.; In Chapter 1, a review was given about high-throughput methods that have been developed to date. A brief description of each method was provided, followed by technical advances and applications of each method. We highlight whether or not a method can be used to assay enzymatic activities and catalytic turnovers since this will help to understand the chemical complementation method we developed.; In Chapter 2 through Chapter 4, the development and application of chemical complementation method was described. We first designed and synthesized a dexamethasone (Dex)-methotrexate (Mtx) small molecule (2–11) and showed that it can efficiently dimerize two fusion proteins, LexA fused to Escherichia coli (E. coli) dihydrofolate reductase (DHFR) and B42 fused to rat glucocorticoid receptor (GR), and activate the reporter gene transcription in the yeast three-hybrid system. We then introduced a cephem linker into the Dex-Mtx small molecule. The cephem linker can be hydrolyzed by the P99 cephalosporinase, and once it is hydrolyzed, it triggers the cleavage of the Dex-Cephem-Mtx (3–13) dimerizer. We have showed that with this compound, we can differentiate cells with either an active cephalosporinase, with an inactive cephalosporinase or without any cephalosporinase, demonstrating the principle of the chemical complementation method. We call this method chemical complementation in analogous to genetic complementation because here a chemical small molecule activates the transcription of a reporter gene which complements a knocked out function. At the end, we applied this chemical complementation method to carbohydrate chemistry. Specifically, we chose to detect a Humicola insolens Ce17B glycosynthase activity, which is an enzyme that can catalyze the formation of a β-1,4 glucosidic bond with appropriate glycosyl donor and acceptors. A glycosyl fluoride donor, Mtx-Lac-F (4–17), and a glycosyl acceptor, Dex-Cel (4–4), were synthesized. With these two compounds, we showed that Cel7B glycosynthase activity can be detected using chemical complementation. Both a mock selection and a true library selection were carried out using this method. Active glycosynthases can be isolated from the libraries, and the statistics of mutants isolated agrees well with published data on other glycosythases. This application further demonstrated the generality and power of chemical complementation. |
