title: Directed evolution of a lysosomal enzyme with enhanced activity at neutral pH by mammalian cell-surface display.
abstract: Human beta-glucuronidase, due to low intrinsic immunogenicity in humans, is an attractive enzyme for tumor-specific prodrug activation, but its utility is hindered by low activity at physiological pH. Here we describe the development of a high-throughput screening procedure for enzymatic activity based on the stable retention of fluorescent reaction product in mammalian cells expressing properly folded glycoproteins on their surface. We utilized this procedure on error-prone PCR and saturation mutagenesis libraries to isolate beta-glucuronidase tetramers that were up to 60-fold more active (k(cat)/K(m)) at pH 7.0 and were up to an order of magnitude more effective at catalyzing the conversion of two structurally disparate glucuronide prodrugs to anticancer agents. The screening procedure described here can facilitate investigation of eukaryotic enzymes requiring posttranslational modifications for biological activity.
authors.affiliation: Institute of Biomedical Sciences, Academia Sinica, Taipei 11529, Taiwan.
authors.forename: Kai-Chuan
authors.initials: KC
authors.lastname: Chen
authors.forename: Chia-Hung
authors.initials: CH
authors.lastname: Wu
authors.forename: Chuan-Yuan
authors.initials: CY
authors.lastname: Chang
authors.forename: Wei-Cheng
authors.initials: WC
authors.lastname: Lu
authors.forename: Qingzong
authors.initials: Q
authors.lastname: Tseng
authors.forename: Zeljko M
authors.initials: ZM
authors.lastname: Prijovich
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Schechinger
authors.forename: Yen-Chywan
authors.initials: YC
authors.lastname: Liaw
authors.forename: Yu-Lin
authors.initials: YL
authors.lastname: Leu
authors.forename: Steve R
authors.initials: SR
authors.lastname: Roffler
mesh_terms: D000818:Animals; D014644:Genetic Variation; D005966:Glucuronidase / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008247:Lysosomes / Q000201:enzymology* / Q000235:genetics; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D008958:Models, Molecular; D015394:Molecular Structure; D011355:Prodrugs / Q000378:metabolism; D015202:Protein Engineering; D015854:Up-Regulation
chemical_list: D008565:Membrane Proteins; D011355:Prodrugs; D005966:Glucuronidase