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: Feng Cheng, Tsvetan Kardashliev, Christian Pitzler, Aamir Shehzad, Hongqi Lue, Jürgen Bernhagen, Leilei Zhu, Ulrich Schwaneberg
• Journal: ACS synthetic biology (ACS Synth Biol) Vol. 4 Issue 7 Pg. 768-75 (Jul 17 2015) ISSN: 2161-5063 [Electronic] United States
• PMID: 25658761 (Publication Type: Letter, Research Support, Non-U.S. Gov't)

Chemical References

• Green Fluorescent Proteins
• Arginine
• Hydrolases
• arginine deiminase

Topics

• Arginine (metabolism)
• Cell Line, Tumor
• Directed Molecular Evolution
• Flow Cytometry
• Green Fluorescent Proteins (genetics, metabolism)
• High-Throughput Screening Assays
• Humans
• Hydrolases (genetics, metabolism)
• Melanoma (metabolism, pathology)
• Mutation
• Protein Engineering
• Pseudomonas (enzymology)
• Skin Neoplasms (metabolism, pathology)