Directed arginine deiminase evolution for efficient inhibition of arginine-auxotrophic melanomas
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| 024 | 7 | 0 | |a 10.1007/s00253-014-5985-z |2 doi |
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| 245 | 0 | 0 | |a Directed arginine deiminase evolution for efficient inhibition of arginine-auxotrophic melanomas |h [Elektronische Daten] |c [Feng Cheng, Leilei Zhu, Hongqi Lue, Jürgen Bernhagen, Ulrich Schwaneberg] |
| 520 | 3 | |a 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). | |
| 540 | |a Springer-Verlag Berlin Heidelberg, 2014 | ||
| 690 | 7 | |a Directed evolution |2 nationallicence | |
| 690 | 7 | |a Arginine-auxotrophic melanoma |2 nationallicence | |
| 690 | 7 | |a Arginine deiminase |2 nationallicence | |
| 690 | 7 | |a Lower K M value |2 nationallicence | |
| 690 | 7 | |a Antiproliferation activity |2 nationallicence | |
| 700 | 1 | |a Cheng |D Feng |u Lehrstuhl für Biotechnologie, RWTH Aachen University, Worringerweg 3, 52074, Aachen, Germany |4 aut | |
| 700 | 1 | |a Zhu |D Leilei |u Lehrstuhl für Biotechnologie, RWTH Aachen University, Worringerweg 3, 52074, Aachen, Germany |4 aut | |
| 700 | 1 | |a Lue |D Hongqi |u Institute of Biochemistry and Molecular Cell Biology, University Hospital Aachen, RWTH Aachen University, Pauwelsstraße 30, 52074, Aachen, Germany |4 aut | |
| 700 | 1 | |a Bernhagen |D Jürgen |u Institute of Biochemistry and Molecular Cell Biology, University Hospital Aachen, RWTH Aachen University, Pauwelsstraße 30, 52074, Aachen, Germany |4 aut | |
| 700 | 1 | |a Schwaneberg |D Ulrich |u Lehrstuhl für Biotechnologie, RWTH Aachen University, Worringerweg 3, 52074, Aachen, Germany |4 aut | |
| 773 | 0 | |t Applied Microbiology and Biotechnology |d Springer Berlin Heidelberg |g 99/3(2015-02-01), 1237-1247 |x 0175-7598 |q 99:3<1237 |1 2015 |2 99 |o 253 | |
| 856 | 4 | 0 | |u https://doi.org/10.1007/s00253-014-5985-z |q text/html |z Onlinezugriff via DOI |
| 898 | |a BK010053 |b XK010053 |c XK010000 | ||
| 900 | 7 | |a Metadata rights reserved |b Springer special CC-BY-NC licence |2 nationallicence | |
| 908 | |D 1 |a research-article |2 jats | ||
| 949 | |B NATIONALLICENCE |F NATIONALLICENCE |b NL-springer | ||
| 950 | |B NATIONALLICENCE |P 856 |E 40 |u https://doi.org/10.1007/s00253-014-5985-z |q text/html |z Onlinezugriff via DOI | ||
| 950 | |B NATIONALLICENCE |P 700 |E 1- |a Cheng |D Feng |u Lehrstuhl für Biotechnologie, RWTH Aachen University, Worringerweg 3, 52074, Aachen, Germany |4 aut | ||
| 950 | |B NATIONALLICENCE |P 700 |E 1- |a Zhu |D Leilei |u Lehrstuhl für Biotechnologie, RWTH Aachen University, Worringerweg 3, 52074, Aachen, Germany |4 aut | ||
| 950 | |B NATIONALLICENCE |P 700 |E 1- |a Lue |D Hongqi |u Institute of Biochemistry and Molecular Cell Biology, University Hospital Aachen, RWTH Aachen University, Pauwelsstraße 30, 52074, Aachen, Germany |4 aut | ||
| 950 | |B NATIONALLICENCE |P 700 |E 1- |a Bernhagen |D Jürgen |u Institute of Biochemistry and Molecular Cell Biology, University Hospital Aachen, RWTH Aachen University, Pauwelsstraße 30, 52074, Aachen, Germany |4 aut | ||
| 950 | |B NATIONALLICENCE |P 700 |E 1- |a Schwaneberg |D Ulrich |u Lehrstuhl für Biotechnologie, RWTH Aachen University, Worringerweg 3, 52074, Aachen, Germany |4 aut | ||
| 950 | |B NATIONALLICENCE |P 773 |E 0- |t Applied Microbiology and Biotechnology |d Springer Berlin Heidelberg |g 99/3(2015-02-01), 1237-1247 |x 0175-7598 |q 99:3<1237 |1 2015 |2 99 |o 253 | ||