The Expression, Purification And Activity Analysis Of Francisella Tularensis Citrulline Ureidase In Escherichia Coli

Citrulline has been proposed as a significant marker for clinical diagnosis. Current methods for citrulline measurement are difficult to perform in practice. Citrulline ureidase (CTU, EC3.5.1.20) from pathogen bacteria Francisella tularensis catalizes citrulline into ornithine, CO2 and NH3, which is much easier to measure. Citrulline ureidase play an extremely important role in rapid detection of citrulline content in human body. Therefore, in this study, we used Escherichia coli to express the protein CTU783, CTU858 and OCT (Ornithine carbamoyl transferase) which maybe have the activity of citrulline ureidase. The pET-22b (+) vector was used and expressed in periplasmic space of E.coli. This plasmid has a His-tage which is easy for purification. Three vectors were successfully constructed and transformed into host strain BL21 (DE3). Three recombinant proteins were expressed and purified successfully.In order to improve the expression level and solubility of the target protein, multiple conditions had been studied. The result showed that when the recombinant strain was cultured at 35 ℃, pH 7.5, with the addition of 1% sorbitol and 0.25 mM IPTG and induced for 6 h, the expression of soluble CTU858 protein,the expression was significantly increased.Because the target protein has a His-tag, so the Ni2+ metal chelate affinity chromatography had been used to separate the target protein, and then get the protein liquid by gradient dialysis and ultrafiltration concentrated. The enzyme activity was measured in first by thin layer chromatography of recombinant proteins, and CTU858 had been found showed activity of citrulline ureidase. Further PITC pre-column derivatization HPLC was used for the quantitative assay of activity of soluble CTU858, CTU783 and inclusion body CTU858 of activity. The soluble CTU858 showed citrulline ureidase activity, the enzyme concentration was 6080 U/L and the specific activity was 12.02 U/mg. The partial inclusion bodies of CTU858 recovered the activity. The CTU783 did not show the activity of citrulline.Citrulline ureidase and glutamate dehydrogenase coupling system and affecting factors have been studied. The main factors affecting the enzymatic activity include substrate concentration, pH, temperature and GLDH. The coupled enzyme method is used to study the feasibility of detection of citrulline in humanbody. The findings of the study will be useful for development of enzymatic measurement method of citrulline.