caa a22 4500 605501661 CHVBK 20210128100602.0 cr unu---uuuuu 210128e20150201xx s 000 0 eng 10.1007/s00253-014-5985-z doi (NATIONALLICENCE)springer-10.1007/s00253-014-5985-z Directed arginine deiminase evolution for efficient inhibition of arginine-auxotrophic melanomas [Elektronische Daten] [Feng Cheng, Leilei Zhu, Hongqi Lue, Jürgen Bernhagen, Ulrich Schwaneberg] Arginine deiminase (ADI) is a therapeutic protein for cancer therapy of arginine-auxotrophic tumors. However, ADI's application as anticancer drug is hampered by its low activity for arginine under physiological conditions mainly due to its high "K M” (S0.5) values which are often 1 magnitude higher than the arginine concentration in blood (0.10-0.12mM arginine in human plasma). Previous evolution campaigns were directed by us with the aim of boosting activity of PpADI (ADI from Pseudomonas plecoglossicida, k cat = 0.18s−1; S 0.5 = 1.30mM), and yielded variant M6 with slightly reduced S 0.5 values and enhanced k cat (S 0.5 = 0.81mM; k cat = 11.64s−1). In order to further reduce the S 0.5 value and to increase the activity of PpADI at physiological arginine concentration, a more sensitive screening system based on ammonia detection in 96-well microtiter plate to reliably detect ≥0.005mM ammonia was developed. After screening ~5,500 clones with the ammonia detection system (ADS) in two rounds of random mutagenesis and site-directed mutagenesis, variant M19 with increased k cat value (21.1s−1; 105.5-fold higher compared to WT) and reduced S 0.5 value (0.35mM compared to 0.81mM (M6) and 1.30mM (WT)) was identified. Improved performance of M19 was validated by determining IC50 values for two melanoma cell lines. The IC50 value for SK-MEL-28 dropped from 8.67 (WT) to 0.10 (M6) to 0.04μg/mL (M19); the IC50 values for G361 dropped from 4.85 (WT) to 0.12 (M6) to 0.05μg/mL (M19). Springer-Verlag Berlin Heidelberg, 2014 Directed evolution nationallicence Arginine-auxotrophic melanoma nationallicence Arginine deiminase nationallicence Lower K M value nationallicence Antiproliferation activity nationallicence Cheng Feng Lehrstuhl für Biotechnologie, RWTH Aachen University, Worringerweg 3, 52074, Aachen, Germany aut Zhu Leilei Lehrstuhl für Biotechnologie, RWTH Aachen University, Worringerweg 3, 52074, Aachen, Germany aut Lue Hongqi Institute of Biochemistry and Molecular Cell Biology, University Hospital Aachen, RWTH Aachen University, Pauwelsstraße 30, 52074, Aachen, Germany aut Bernhagen Jürgen Institute of Biochemistry and Molecular Cell Biology, University Hospital Aachen, RWTH Aachen University, Pauwelsstraße 30, 52074, Aachen, Germany aut Schwaneberg Ulrich Lehrstuhl für Biotechnologie, RWTH Aachen University, Worringerweg 3, 52074, Aachen, Germany aut Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/3(2015-02-01), 1237-1247 0175-7598 99:3<1237 2015 99 253 https://doi.org/10.1007/s00253-014-5985-z text/html Onlinezugriff via DOI BK010053 XK010053 XK010000 Metadata rights reserved Springer special CC-BY-NC licence nationallicence 1 research-article jats NATIONALLICENCE NATIONALLICENCE NL-springer NATIONALLICENCE 856 40 https://doi.org/10.1007/s00253-014-5985-z text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Cheng Feng Lehrstuhl für Biotechnologie, RWTH Aachen University, Worringerweg 3, 52074, Aachen, Germany aut NATIONALLICENCE 700 1- Zhu Leilei Lehrstuhl für Biotechnologie, RWTH Aachen University, Worringerweg 3, 52074, Aachen, Germany aut NATIONALLICENCE 700 1- Lue Hongqi Institute of Biochemistry and Molecular Cell Biology, University Hospital Aachen, RWTH Aachen University, Pauwelsstraße 30, 52074, Aachen, Germany aut NATIONALLICENCE 700 1- Bernhagen Jürgen Institute of Biochemistry and Molecular Cell Biology, University Hospital Aachen, RWTH Aachen University, Pauwelsstraße 30, 52074, Aachen, Germany aut NATIONALLICENCE 700 1- Schwaneberg Ulrich Lehrstuhl für Biotechnologie, RWTH Aachen University, Worringerweg 3, 52074, Aachen, Germany aut NATIONALLICENCE 773 0- Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/3(2015-02-01), 1237-1247 0175-7598 99:3<1237 2015 99 253