E. coli BL21(DE3) competent cells were transformed with pET30a-UreB/PLA1/Omp6 and inoculated in LB broth with kanamycin (50 μg/mL). All three expression was induced by addition of Isopropyl β-d-1-Thiogalactopyranoside (IPTG) after the optical density (OD600) had reached 0.6. The gradient of IPTG concentration and induced temperature were explored. After 6 h the whole proteins were extracted from the collected cells by One Step Bacteria Active Protein Extraction Kit (Sangon Biotech, China) and analyzed by Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). The soluble histidin-tagged proteins were purified using affinity chromatography (Ni-IDA-Sefinose Column, Sangon Biotech, China).

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