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).

Rational surface engineering of an arginine deiminase (an antitumor enzyme) for increased PEGylation efficiency

“…Absorbance was measured at 490 nm using an MTP reader (Tecan Sunrise; Tecan Group AG, Zürich, Switzerland). The ammonia‐detection system was used for detection of the kinetic data of non‐PEGylated and PEGylated PpADI variants as described in the previously published paper (Cheng, Kardashliev, et al, ).…”

“…In our previous work, a PpADI variant M31 was identified with significantly improved activity after several rounds of directed evolution campaigns with biosensor‐based high‐throughput screening (F. Cheng, Kardashliev, et al, ; F. Cheng, Tang, & Kardashliev, ). The in vitro tumor cell line tests showed that M31 displayed the dramatically reduced IC 50 values against melanoma cell lines SK‐MEL‐28 (0.02 µg/ml; 0.24% of WT) and G361 (0.02 µg/ml; 0.45% of WT).…”

Transcription Factor‐Based Biosensors in High‐Throughput Screening: Advances and Applications

“…Such concentrations are one order of magnitude higher than those of many metabolites in living cells or in blood plasma (e.g., in blood, [arginine] ≈100 μM) . A ligand‐mediated eGFP‐expression system (LiMEx) was developed by Cheng et al as a novel flow cytometry‐based screening platform that relies on a competitive conversion/binding of arginine between arginine deiminase and an arginine‐binding repressor protein . Unlike production‐driven detection systems (Figure A), the competitive screening platform allows to evolve enzymes toward more efficient operation at or below physiological substrate concentrations (Figure B).…”

“…Other therapeutic enzymes could be conceptually evolved via LiMEx. For instance, asparaginase, a drug for treatment of leukemia, could be evolved by an “Asp‐LiMEx system” based on a competition between L ‐asparagine operon repressor (AnsR) and asparaginase toward asparagine (physiological concentration ≈0.1 mM) …”

Directed evolution 2.0: improving and deciphering enzyme properties

“…Nowadays, protein engineering by directed evolution has become a standard method to tailor enzyme properties in academia and industry (mainly for biocatalysis 8 purposes). Novel application fields in material science 9 and medical science [10][11][12] will likely lead to a further growth in directed evolution reports despite that the development time for improving enzymes by directed evolution are often still too long to be implemented as a standard operation in process development. Till today most directed evolution campaigns are performed in a traditional manner, in which usually random mutagenesis libraries are generated through epPCR with low mutations frequencies (1 to 3 mutations per 1000 bp) and screened (MTP format; often 1000-2000 variants) in order to obtained improved variants.…”