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@PHDTHESIS{Zhu:63710,
author = {Zhu, Leilei},
othercontributors = {Schwaneberg, Ulrich},
title = {{D}irected evolution of arginine deiminase ({ADI}) for
anti-tumor application},
address = {Aachen},
publisher = {Publikationsserver der RWTH Aachen University},
reportid = {RWTH-CONV-125135},
pages = {IX, 98 : Ill., graph. Darst.},
year = {2010},
note = {Aachen, Techn. Hochsch., Diss., 2010},
abstract = {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 $12.8\%$ was
obtained, which is appropriate for the library screening.
With the established microtiter plate assay system, for the
first time, we reengineered ADI for improved activity at
physiological pH (pH 7.4) with higher potency in medical
application. After one site directed mutagenesis and one
round of epPCR library screening, variant M2
(K5T/D44E/H404R) was obtained. M2 shows 4-fold improved kcat
value than the PpADI wild type, a shifted pH optimum at pH
7.0 (by 0.5 pH unit), and however an increased Km value,
3.6-fold under assay conditions (0.5 M sodium phosphate
buffer). Low Km (S0.5) value for ADI is an important factor
for efficient consume of arginine in plasma because of the
low arginine concentration in human plasma (100-120 µM).
Therefore after our proof of concept of reengineering PpADI
by directed evolution, we aim to decrease the Km value of
PpADI towards arginine and improve the kcat furthermore
thereby improve the efficiency of arginine depletion by ADI.
The established citrulline colorimetric assay in 96-well
microtiter plate is improved to reflect application
conditions: PBS buffer and 1 mM arginine were used in
screening system. After the second round of epPCR library
and site directed mutagenesis, variant M5
(K5T/D38H/D44E/A128T/H404R) and M6
(K5T/D38H/D44E/A128T/E296K/H404R) were generated: at pH 7.4
(PBS buffer), the S0.5 value decrease from 2.01 mM (parent
M3, K5T/D44E/A128T/H404R) to 1.48 mM (M5) and 0.81 mM (M6).
Under physiological pH in PBS buffer, the S0.5 value of M6
(0.81 mM) is lower than that of PpADI wild type (1.30 mM);
the kcat value improved from 0.18 s-1 (PpADI wild type) to
17.56 s-1 (M5, 97.6-folds) and 11.64 s-1 (M6, 64.7-fold).},
keywords = {Gerichtete Evolution (SWD)},
cin = {162610 / 160000},
ddc = {570},
cid = {$I:(DE-82)162610_20140620$ / $I:(DE-82)160000_20140620$},
shelfmark = {WF 9720},
typ = {PUB:(DE-HGF)11},
urn = {urn:nbn:de:hbz:82-opus-33679},
url = {https://publications.rwth-aachen.de/record/63710},
}
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