Expression,purification and activity of Arthrobacter globiformis uricase

Objective To express Arthrobacter globiformis uricase(Uri) in E. coli,purify the expressed product and determine its activity. Methods According to the codon bias of E. coli,and the minimization principle of free energy of local secondary structure of original sequence for prokaryotic translation,uri gene was designed,amplified by PCR and cloned into vector pET43.1a. The constructed recombinant plasmid pET43.1a-uri was transformed to E. coli BL21-CodonPlus(DE3)-RIPL for expression under induction of IPTG. The expressed product was purified by ammonium sulfate precipitation followed by DEAE agarose gel chromatography,and analyzed for purity by SDS-PAGE. Then purified product was determined for protease activity according to the instruction of Uri,and the temperature and pH value for determination were optimized. Results Restriction analysis and sequencing proved that recombinant plasmid pET43.1a-uri was constructed correctly. The expressed recombinant uricase,with a relative molecular mass of about 33 000,existed in a soluble form,contained about 40% of total cellular protein,and reached a purity of more than 90% after purification, of which the protease activity was 13. 2 U / mg. The optimal temperature and pH value for determination of protease activity were 40 ℃ and 9 respectively. Conclusion The uri gene was successfully expressed in E. coli,and highly purified Uri was obtained,of which the enzymatic properties were basically consistent with those of natural A. globiformis Uri. It laid a foundation of large-scale and stable production of Uri and development of detection kit.