Open AccessDiscovery of human-like L-asparaginases with potential clinical use by directed evolution
Author(s) -
Coraline Rigouin,
Hien Anh Thi Nguyen,
Amanda M. Schalk,
A. Lavie
Publication year - 2017
Publication title -
scientific reports
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.24
H-Index - 213
ISSN - 2045-2322
DOI - 10.1038/s41598-017-10758-4
Subject(s) - dna shuffling , asparagine , asparaginase , immunogenicity , biology , amino acid , computational biology , biochemistry , genetics , gene , directed evolution , lymphoblastic leukemia , leukemia , mutant , antigen
L-asparaginase is a chemotherapy drug used to treat acute lymphoblastic leukemia (ALL). The main prerequisite for clinical efficacy of L-asparaginases is micromolar K M for asparagine to allow for complete depletion of this amino acid in the blood. Since currently approved L-asparaginases are of bacterial origin, immunogenicity is a challenge, which would be mitigated by a human enzyme. However, all human L-asparaginases have millimolar K M for asparagine. We recently identified the low K M guinea pig L-asparaginase (gpASNase1). Because gpASNase1 and human L-asparaginase 1 (hASNase1) share ~70% amino-acid identity, we decided to humanize gpASNase1 by generating chimeras with hASNase1 through DNA shuffling. To identify low K M chimeras we developed a suitable bacterial selection system ( E. coli strain BW5Δ). Transforming BW5Δ with the shuffling libraries allowed for the identification of several low K M clones. To further humanize these clones, the C-terminal domain of gpASNase1 was replaced with that of hASNase1. Two of the identified clones, 63 N -h C and 65 N -h C , share respectively 85.7% and 87.1% identity with the hASNase1 but have a K M similar to gpASNase1. These clones possess 100–140 fold enhanced catalytic efficiency compared to hASNase1. Notably, we also show that these highly human-like L-asparaginases maintain their in vitro ALL killing potential.