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A Competitive Flow Cytometry Screening System for Directed Evolution of Therapeutic Enzyme
Feng Cheng1, Tsvetan Kardashliev1,2, Christian Pitzler1
1†Lehrstuhl für Biotechnologie, RWTH Aachen University, Worringerweg 3, 52074 Aachen, Germany.
ACS Synthetic Biology
|February 7, 2015
Summary
A novel screening platform, LiMEx, enables enzyme evolution for enhanced therapeutic efficacy. A variant of arginine deiminase (ADI) was developed to effectively inhibit melanoma tumor growth.
Area of Science:
- Biotechnology
- Enzyme Engineering
- Flow Cytometry
Background:
- Developed a ligand-mediated eGFP-expression system (LiMEx) for enzyme evolution.
- LiMEx utilizes a competitive binding assay for arginine, enabling screening under physiological conditions.
- This platform overcomes limitations of product-driven detection systems.
Discussion:
- Evolved arginine deiminase (ADI) using LiMEx for improved anticancer activity.
- Screened approximately 8.2 x 10^6 clones through iterative epPCR libraries.
- Identified a variant, M31, with significantly reduced substrate affinity (S0.5) and enhanced activity at physiological arginine concentrations.
Key Insights:
- The evolved PpADI variant M31 exhibits a 7-fold lower S0.5 value (0.17 mM vs. 1.23 mM) compared to wild-type.
- M31 demonstrates detectable activity at physiological arginine levels, unlike wild-type ADI.
- M31 showed potent inhibition against SK-MEL-28 and G361 melanoma cell lines with an IC50 of 0.02 μg/mL.
Outlook:
- LiMEx platform holds potential for evolving enzymes for various therapeutic applications.
- Further studies can explore optimizing ADI variants for broader anticancer efficacy.
- This approach can accelerate the development of novel enzyme-based therapeutics.
