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The expression, purification and activity analysis of Francisella tularensis citrulline ureidase in Escherichia coli
Tingheng Zhu, Sanyu Fang, Weixia Wang, Kun Wang, Zhifeng Cui, Changchun Wang
Language English Country Czech Republic
Document type Research Support, Non-U.S. Gov't
- MeSH
- Bacteria MeSH
- Bacteriological Techniques methods utilization MeSH
- Chromatography, Affinity methods utilization MeSH
- Citrulline * isolation & purification metabolism MeSH
- Enzyme Assays methods utilization MeSH
- Escherichia coli enzymology immunology isolation & purification MeSH
- Francisella tularensis * enzymology isolation & purification metabolism MeSH
- Hydrolases isolation & purification MeSH
- Ornithine * isolation & purification metabolism MeSH
- Recombinant Proteins genetics isolation & purification metabolism MeSH
- Sorbitol diagnostic use MeSH
- Statistics as Topic MeSH
- Tularemia diagnosis etiology microbiology MeSH
- Urease isolation & purification metabolism MeSH
- Chromatography, High Pressure Liquid methods utilization MeSH
- Publication type
- Research Support, Non-U.S. Gov't MeSH
Citrulline ureidase (CTU, EC3.5.1.20) degrades citrulline into ornithine, carbon dioxide, and ammonia. Here, we present the report on expression of recombinant CTU in Escherichia coli. The soluble and active recombinant CTU was expressed in the periplasmic space with the vector pET-22b and the His-tagged CTU was purified with Ni-Affinity Chromatography. The yield of soluble recombinant protein was significantly increased when 1% sorbitol was supplemented in medium. By using phenylisothiocyanate (PITC) pre-column derivatization HPLC, the enzyme activity of recombinant CTU was determined via measuring of the substrate citrulline and the corresponding products. Our results could be useful in the study of CTU biochemical characteristics, enzymatic preparation of ornithine and development of an enzymatic detection method of citrulline.
China National Rice Research Institute Hangzhou 310006 China
College of Chemistry and Life Sciences Zhejiang Normal University Jinhua 321004 China
References provided by Crossref.org
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- $a Citrulline ureidase (CTU, EC3.5.1.20) degrades citrulline into ornithine, carbon dioxide, and ammonia. Here, we present the report on expression of recombinant CTU in Escherichia coli. The soluble and active recombinant CTU was expressed in the periplasmic space with the vector pET-22b and the His-tagged CTU was purified with Ni-Affinity Chromatography. The yield of soluble recombinant protein was significantly increased when 1% sorbitol was supplemented in medium. By using phenylisothiocyanate (PITC) pre-column derivatization HPLC, the enzyme activity of recombinant CTU was determined via measuring of the substrate citrulline and the corresponding products. Our results could be useful in the study of CTU biochemical characteristics, enzymatic preparation of ornithine and development of an enzymatic detection method of citrulline.
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