Directed evolution of a lysosomal enzyme with enhanced activity at neutral pH by mammalian cell-surface display.
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.
Chen
Kai-Chuan
KC
Institute of Biomedical Sciences, Academia Sinica, Taipei 11529, Taiwan.
Wu
Chia-Hung
CH
Chang
Chuan-Yuan
CY
Lu
Wei-Cheng
WC
Tseng
Qingzong
Q
Prijovich
Zeljko M
ZM
Schechinger
Wolfgang
W
Liaw
Yen-Chywan
YC
Leu
Yu-Lin
YL
Roffler
Steve R
SR
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
D008565:Membrane Proteins; D011355:Prodrugs; D005966:Glucuronidase