Cloning, purification, and partial characterization of Bacillus subtilis urate oxidase expressed in Escherichia coli

Abstract

Urate oxidase (EC 1.7.3.3) is an enzyme involved in purine metabolism which is used in the treatment of gout and as diagnostic reagent for detection of uric acid. In order to produce this enzyme in large quantities for biotechnological purposes, the gene coding for the Bacillus subtilis urate oxidase was cloned and heterologously expressed in Escherichia coli. Time course induction in E. coli showed an induced protein with an apparent molecular mass of approximately 60 kDa. Soluble recombinant enzyme was purified in a single-step procedure using Ni-NTA column. The enzyme was purified 2.1-fold with a yield of 56% compared to the crude extract. MALDI-TOF analysis revealed an ion with a mass of 58675 Da which is in agreement with the expected mass of the recombinant protein. The purified enzyme showed an optimal pH and temperature of 8.0 and 37 degrees C, respectively, and retained 90% of its activity after 72 hours of incubation at -20 degrees C and 4 degrees C.

Figure 1

Small-scale expression and time course study of urate oxidase expression in E. coli. Comassie Blue stained 12% SDS-PAGE (a) and respective Western blotting analysis (b) of urate oxidase induction in E. coli. Lane M, protein BenchMarker (Invitrogen); Lane 1, supernatant of uninduced cells; Lanes 2–6, supernatant of induced cells after 15, 30, 60, 90, and 120 minutes, respectively. The arrow indicates the position of the band corresponding to recombinant urate oxidase (rUox).

Figure 2

Recombinant enzyme purification. Fractions collected before and after Ni-NTA chromatography were visualized on 12% SDS-PAGE. Lane M, protein BenchMarker (Invitrogen); Lane 1, crude extract of uninduced E. coli cells; Lane 2, crude cell extract after induction for 3 hours; Lane 3, cellular extract (input); Lane 4, flow-through; Lane 5, washing step (20 mM imidazole); Lane 6, purified urate oxidase (~30.5 μg) after elution with 200 mM imidazole.

Figure 3

Mass spectometry. MALDI-TOF/MS spectrum of recombinant urate oxidase using sinapinic acid as matrix showed two related ions for the same polypeptide chain, M+2H⁺ = 29338.1 Da and M+H⁺ = 58675 Da.

Figure 4

Effect of pH and temperature on enzyme activity. Purified urate oxidase was incubated under different pHs (a) and temperatures (b) and assayed as described in Section 2.

Figure 5

Thermal stability. Purified enzyme was pre-incubated at −20°C, 4°C and 37°C for the indicated times, and assayed at 37°C. Residual activity corresponds to the percentage of activity determined for the nonincubated samples.