Purification and characterization of recombinant Bacillus subtilis 168 catalase using a basic polypeptide from ribosomal protein L2 |
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| Institution: | 1. Department of Cellular Biology and Pharmacology, Herbert Wertheim College of Medicine, Florida International University, Miami, FL 33199, USA;2. Department of Occupational and Environmental Health, Nagoya University Graduate School of Medicine, Nagoya, Aichi 466-8550, Japan;3. SER-CAT and the Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA 30602, USA;4. Molecular Biophysics and Integrated Bioimaging, Lawrence Berkeley Laboratory, Berkeley Center for Structural Biology, Berkeley, CA 94720, USA;1. Department of Pathophysiology, Key Laboratory of Cardiovascular Disease and Molecular Intervention, Nanjing Medical University, Nanjing, China;2. State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing, China;3. Jiangsu Institute of Nuclear Medicine, Wuxi, China;4. Department of Gastroenterology, Second Hospital Affiliated to Nanjing Medical University, Nanjing, China;5. Department of Endocrinology, Nanjing First Hospital, Nanjing Medical University, Nanjing, China |
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| Abstract: | An efficient purification system for purifying recombinant Bacillus subtilis 168 catalase (KatA) expressed in Escherichia coli was developed. The basic region containing 252–273 amino acids derived from E. coli ribosomal protein L2 was used as an affinity tag while the small ubiquitin-like modifier (SUMO) was introduced as one specific protease cleavage site between the target protein and the purification tags. L2 (252–273)–SUMO fusion protein purification method can be effectively applied to purify the recombinant catalase using cation exchange resin. This purification procedure was used to purify the KatA and achieved a purification fold of 30.5, a specific activity of 48,227.2 U/mg and an activity recovery of 74.5%. The enzyme showed a Soret peak at 407 nm. The enzyme kept its activity between pH 5 and 10 and between 30 °C and 60 °C, with the highest activity at pH 8.0 and 37 °C. The enzyme displayed an apparent Km of 39.08 mM for hydrogen peroxide. These results agree well with the previous reports about B. subtilis catalase. L2 (252–273)–SUMO fusion protein purification technique provides a novel and effective fusion expression system for the production of recombinant proteins. |
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