title: A Competitive Flow Cytometry Screening System for Directed Evolution of Therapeutic Enzyme.
abstract: A ligand-mediated eGFP-expression system (LiMEx) was developed as a novel flow cytometry based screening platform that relies on a competitive conversion/binding of arginine between arginine deiminase and arginine repressor. Unlike product-driven detection systems, the competitive screening platform allows to evolve enzymes toward efficient operation at low substrate concentrations under physiological conditions. The principle of LiMEx was validated by evolving arginine deiminase (ADI, an anticancer therapeutic) for stronger inhibition of tumor growth. After screening of ∼8.2 × 10(6) clones in three iterative rounds of epPCR libraries, PpADI (ADI from Pseudomonas plecoglossicida) variant M31 with reduced S0.5 value (0.17 mM compared to 1.23 mM (WT)) and, importantly, increased activity at physiological arginine concentration (M31:6.14 s(-1); WT: not detectable) was identified. Moreover, M31 showed a significant inhibitory effect against SK-MEL-28 and G361 melanoma cell lines. (IC50 = 0.02 μg/mL for SK-MEL-28 and G361).
authors: †Lehrstuhl für Biotechnologie, RWTH Aachen University, Worringerweg 3, 52074 Aachen, Germany.
authors: Feng
authors: F
authors: Cheng
authors: †Lehrstuhl für Biotechnologie, RWTH Aachen University, Worringerweg 3, 52074 Aachen, Germany.|‡DWI-Leibniz Institute for Interactive Materials, Forckenbeckstraße 50, 52056 Aachen, Germany.
authors: Tsvetan
authors: T
authors: Kardashliev
authors: †Lehrstuhl für Biotechnologie, RWTH Aachen University, Worringerweg 3, 52074 Aachen, Germany.
authors: Christian
authors: C
authors: Pitzler
authors: †Lehrstuhl für Biotechnologie, RWTH Aachen University, Worringerweg 3, 52074 Aachen, Germany.
authors: Aamir
authors: A
authors: Shehzad
authors: §Institute of Biochemistry and Molecular Cell Biology, University Hospital Aachen, RWTH Aachen University, Pauwelsstraße 30, 52074 Aachen, Germany.
authors: Hongqi
authors: H
authors: Lue
authors: §Institute of Biochemistry and Molecular Cell Biology, University Hospital Aachen, RWTH Aachen University, Pauwelsstraße 30, 52074 Aachen, Germany.
authors: Jürgen
authors: J
authors: Bernhagen
authors: †Lehrstuhl für Biotechnologie, RWTH Aachen University, Worringerweg 3, 52074 Aachen, Germany.
authors: Leilei
authors: L
authors: Zhu
authors: †Lehrstuhl für Biotechnologie, RWTH Aachen University, Worringerweg 3, 52074 Aachen, Germany.|‡DWI-Leibniz Institute for Interactive Materials, Forckenbeckstraße 50, 52056 Aachen, Germany.
authors: Ulrich
authors: U
authors: Schwaneberg
mesh_terms: D001120:Arginine / Q000378:metabolism; D045744:Cell Line, Tumor; D019020:Directed Molecular Evolution; D005434:Flow Cytometry; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D057166:High-Throughput Screening Assays; D006801:Humans; D006867:Hydrolases / Q000235:genetics / Q000378:metabolism*; D008545:Melanoma / Q000378:metabolism / Q000473:pathology; D009154:Mutation; D015202:Protein Engineering; D011549:Pseudomonas / Q000201:enzymology; D012878:Skin Neoplasms / Q000378:metabolism / Q000473:pathology
chemical_list: D049452:Green Fluorescent Proteins; D001120:Arginine; D006867:Hydrolases; C019723:arginine deiminase
keywords: LiMEx; arginine deiminase; competitive screening system; directed evolution; protein engineering; synthetic biology