High-level production of uricase containing keto functional groups for site-specific PEGylation

Abstract We describe an E. coli -based optimized system for the production of uricase with keto functional groups incorporated efficiently and site-specifically. In the process, the orthogonal suppressor tRNA/aminoacyl-tRNA synthetase (aaRS) pair specific for p -acetylphenylalanine ( p AcF) was optimized to be effective at p AcF incorporation, showing no toxicity to the host cells. The efficiency of p AcF incorporation was further improved by coupling five copies of the T-stem mutant suppressor tRNA gene omitted the 3′ terminal CCA with two constitutive copies of the D286R mutant aaRS gene in a single-plasmid construct. To assay the utility of the optimized system, we incorporated p AcF in response to three independent amber nonsense codons (Lys21TAG, Phe170TAG, Lys248TAG) into uricase. Under optimized expression conditions, 24 mg/L mutant uricase was produced, corresponding to 40% of the yield of wild-type uricase (UOXWT). The desired specificity for incorporation of p AcF into uricase was confirmed. Kinetic measurements and spectroscopic study performed by CD did not show any relevant differences in the substrate affinity, the catalytic activity and protein secondary structure between native and mutant uricase. Additionally, the mutant uricase was site-specifically modified with methoxy-PEG-oxyamine (mPEG 5K -ONH 2 ). This efficient system provides reactive handles for a rational PEGylation to manipulate uricase structure and function and will be beneficial for enhancing the incorporation of other unnatural amino acids into proteins.