Cloning And Expression Of Arginine Deiminase Gene In E.coli

As one of the nonprotein amino acids, citrulline plays an important role in the urea cycle. Citrulline also has a lot of important physiological functions in the area such as Pharmaceuticals, cosmetic etc. Arginine deiminase (ADI) is an enzyme which can catalyze the imine hydrolysis of arginine to produce citrulline and ammonia. It is a decline to use ADI in the citrulline production industry. Expressing ADI gene in E.coli is an effective way to get ADI.Firstly, ADI gene was cloned from the DNA of Pseudomonas putida by PCR, then the cloned gene was constructed with plasmid pET-30a and pBV220 and transformed into E. coli BL21(DE3) and JM109. The two recombinant strains were named as BL21(DE3)(pET-30a-cit) and JM109(pBV220-cit) respectively. The DNA sequence of cloned gene had 10% difference from that of Pseudomonas putida ATCC 4359 in GenBank, while the amino acids sequence of cloned gene was the same with Pseudomonas putida ATCC 4359.Under the induction of 0.1% IPTG, the expression of BL21(DE3)(pET-30a-cit) showed 235.7 U/mL enzyme activity. However, the ADI enzyme activity of JM109(pBV220-cit) had not been detected. The result of SDS-PAGE showed that the molecular weight of cloned ADI was about 43kDa.