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

. A p15A-based single-plasmid system compatible with most E. coli expression vectors and strains was developed for incorporating p-acetylphenylalanine into uricase at the desired sites with high efficiency and fidelity. Under optimized expression conditions, 24 mg/L mutant uricase was produced, corresponding to 40% of the yield of wild-type uricase. Full-length mutant uricase was only produced in our optimized system when all three orthogonal components were present. 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. [Display omitted] ► A suppressor tRNA was optimized to highly improve the efficiency of pAcF incorporation. ► The efficiency was further enhanced by optimizing suppressor tRNA/aaRS copy number. ► The suppressor tRNA/aaRS pair had no adverse effects on the growth of host cells. ► The versatility of pAC- pAcF produced high-yield of mutant uricase with full activity. ► The keto groups provide reactive handles for a rational PEGylation of uricase. 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 ( pAcF) was optimized to be effective at pAcF incorporation, showing no toxicity to the host cells. The efficiency of pAcF 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 pAcF 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 pAcF 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 react