h1

%0 Thesis
%A Zhu, Leilei
%T Directed evolution of arginine deiminase (ADI) for anti-tumor application
%C Aachen
%I Publikationsserver der RWTH Aachen University
%M RWTH-CONV-125135
%P IX, 98 : Ill., graph. Darst.
%D 2010
%Z Aachen, Techn. Hochsch., Diss., 2010
%X Arginine deiminase (ADI), an arginine-degrading enzyme, is involved in the first step of arginine dihydrolase pathway. It catalyzes the hydrolysis of L-arginine to form citrulline and ammonia. ADI has been studied as a potential cancer therapeutic agent for the arginine-auxotrophic tumors, such as hepatocellular carcinomas (HCC) and melanomas. Furthermore, studies also indicate that ADI is a potential anti-angiogenic agent; therefore it could become a novel anti-cancer drug targeting the neovascularization-related tumors. Studies show ADI is more potent for the treatment of leukemia than L-asparaginase. However, the anti-tumor application of ADI for therapeutic purpose faces considerable challenges, such as, microbial ADI has low activity at physiological pH (7.35 7.45), short circulating half-life ( 5 h) and high antigenicity. Pegylation of ADI improved its efficacy as a clinical drug, including its half-life in serum and antigenicity. The aim of the project is to improve ADI catalytic performance at physiological pH by directed evolution. Protein engineering by rational design and directed evolution offers opportunities to tailor ADI properties to physiological conditions. Directed protein evolution has over the last decades become a versatile and successful approach for tailoring protein properties to industrial demands and for advancing our understanding of structure-function relationships in biocatalysts. Unlike rational design relying on the gathering of extensive structure-function relationships of enzymes, directed evolution is used to reengineer enzyme properties through iterative rounds of diversity generation and function selection for improved variants. In order to improve the ADI activity at physiological pH, we established a directed evolution protocol for this purpose. A microtiter plate (MTP) format colorimetric screening assay based on citrulline detection with diacetyl monoxime (DAM) was developed. Reaction temperature for color development and DAM concentration were optimized to ensure sufficient sensitivity, appropriate linear range and throughput. With the optimized assay and PpADI wild type expressed in E. coli as a model protein, true standard deviation 12.8
%K Gerichtete Evolution (SWD)
%F PUB:(DE-HGF)11
%9 Dissertation / PhD Thesis
%U https://publications.rwth-aachen.de/record/63710