title: Fluorescence-Activated Cell Sorting of Human l-asparaginase Mutant Libraries for Detecting Enzyme Variants with Enhanced Activity.
abstract: Immunogenicity is one of the most common complications occurring during therapy making use of protein drugs of nonhuman origin. A notable example of such a case is bacterial l-asparaginases (L-ASNases) used for the treatment of acute lymphoblastic leukemia (ALL). The replacement of the bacterial enzymes by human ones is thought to set the basis for a major improvement of antileukemic therapy. Recently, we solved the crystal structure of a human enzyme possessing L-ASNase activity, designated hASNase-3. This enzyme is expressed as an inactive precursor protein and post-translationally undergoes intramolecular processing leading to the generation of two subunits which remain noncovalently, yet tightly associated and constitute the catalytically active form of the enzyme. We discovered that this intramolecular processing can be drastically and selectively accelerated by the free amino acid glycine. In the present study, we report on the molecular engineering of hASNase-3 aiming at the improvement of its catalytic properties. We created a fluorescence-activated cell sorting (FACS)-based high-throughput screening system for the characterization of rationally designed mutant libraries, capitalizing on the finding that free glycine promotes autoproteolytic cleavage, which activates the mutant proteins expressed in an E. coli strain devoid of aspartate biosynthesis. Successive screening rounds led to the isolation of catalytically improved variants showing up to 6-fold better catalytic efficiency as compared to the wild-type enzyme. Our work establishes a powerful strategy for further exploitation of the human asparaginase sequence space to facilitate the identification of in vitro-evolved enzyme species that will lay the basis for improved ALL therapy.
authors.affiliation: Enzyme Biochemistry Group, Max-Planck Institute for Biophysical Chemistry , Göttingen, D-37077, Germany.
authors.forename: Christos S
authors.initials: CS
authors.lastname: Karamitros
authors.affiliation: Enzyme Biochemistry Group, Max-Planck Institute for Biophysical Chemistry , Göttingen, D-37077, Germany.
authors.forename: Manfred
authors.initials: M
authors.lastname: Konrad
mesh_terms: D001215:Asparaginase / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism / Q000627:therapeutic use; D005453:Fluorescence; D006801:Humans; D009154:Mutation; D054198:Precursor Cell Lymphoblastic Leukemia-Lymphoma / Q000188:drug therapy
chemical_list: D001215:Asparaginase