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Directed Evolution of an Antitumor Drug (Arginine Deiminase PpADI) for Increased Activity at Physiological pH
Author(s) -
Zhu Leilei,
Tee Kang Lan,
Roccatano Danilo,
Sonmez Burcu,
Ni Ye,
Sun ZhiHao,
Schwaneberg Ulrich
Publication year - 2010
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.200900717
Subject(s) - arginine deiminase , arginine , chemistry , methotrexate , drug , microtiter plate , hccs , biochemistry , pharmacology , chromatography , biology , amino acid , immunology , gene
Arginine deiminase (ADI; EC 3.5.3.6) has been studied as a potential antitumor drug for the treatment of arginine‐auxotrophic tumors, such as hepatocellular carcinomas (HCCs) and melanomas. Studies with human lymphatic leukemia cell lines confirmed that ADI is an antiangiogenic agent for treating leukemia. The main limitation of ADI from Pseudomonas plecoglossicida (PpADI) lies in its pH‐dependent activity profile, its pH optimum is at 6.5. A pH shift from 6.5 to 7.5 results in an approximately 80 % drop in activity. (The pH of human plasma is 7.35 to 7.45.) In order to shift the PpADI pH optimum, a directed‐evolution protocol based on an adapted citrulline‐screening protocol in microtiter‐plate format was developed and validated. A proof of concept for ADI engineering resulted in a pH optimum of pH 7.0 and increased resistance under physiological and slightly alkaline conditions. At pH 7.4, variant M2 (K5T/D44E/H404R) is four times faster than the wild‐type PpADI and retains ∼50 % of its activity relative to its pH optimum, compared to ∼10 % in the case of the wild‐type PpADI.