Study On Heterologous Expression, Purification And Property Of Recombinant Arginine Deiminase

Arginine deiminase (ADI, EC 3.5.3.6), which widely exists in bacteria, achaea and some anaerobic eukarya, is the first enzyme in ADI pathway for arginine degradation. It has been proven to be effective as a potential therapeutic agent for arginine-auxotrophic cancers, leukemia, etc. The recombinant ADI used currently in clinical trials are derived from a parasitic and pathogenic microorganism, M. arginini, it therefore becomes necessary to develop a recombinant ADI with less biosafety issues.In this study, the ADI gene from Pseudomonas plecoglossicida CGMCC2039 was acquired through PCR, sequenced and analyzed. The nucleotide sequence of ADI comprises of an open reading frame of 1,254 bp encoding for 417 amino acids, and deduced protein mass weight is 46.5 kDa. Blast analysis showed 97 % and 85 % identity to ADIs from P. putida and P. aeruginosa.The acquired ADI gene fragment was then cloned to pMD18-T vector. Recombinant expression vector, pET28a-ADI, was constructed through restriction enzyme digestion and ligation, and the construct vector was then transformed into E. coli BL21. The expression of recombinant ADI gene was demonstrated through SDS-PAGE and Western blot analysis, and its enzymatic activity was later confirmed by activity assay. The optimal inducer IPTG concentration, inducing time and temperature were determined to be 0.4 mM, 4 h and 30℃, respectively. The existence of inclusion body was confirmed by SDS-PAGE analysis.The recombinant ADI was one-step purified by using nickel chelated agarose gel affinity chromography. SDS-PAGE anaylysis showed it had a mass weight of approximate 49 kDa as was expected, and the activity of purified enzyme is 4.76 U/mg. The effects of pH, temperature, metal ions and chemicals on the activity of recombinant ADI were investigated, as well as its thermostability and kinetic parameters measurement. The results showed that the optimal pH and reaction temperature were 6.0 and 37℃respectively; metal ions and chemicals have little effects on activity of the recombinant ADI, except for Cu2+; recombinant ADI seems thermo-sensitive, with over 50 % activity lost at 37℃after 100 min; kinetic parameters, Vmax and Km, were determined to be 4.92μmol/min·mg and 96.09 mM, respectively.