Studies On The Secretory Expression Of Arginine Deiminase

Arginine deiminase (ADI; EC 3.4.3.6) is an arginine-degrading enzyme, it hydrolysis arginine to citrulline and ammonia. ADI is a potential cancer therapy agent for the treatment of arginine-auxotrophic tumors (such as hepatocellular carcinomas and melanomas), and it widely exists in bacteria, achaea and some anaerobic eukarya. In our preceding study, a Pseudomonas plecoglossicida GMCC2039 strain with remarkable ADI activity was isolated from nature, and the ADI was expressed in cytoplasma of Escherichia coli BL21, in which over 50% protein was expressed as inclusion body. In order to obtain native recombinant ADI and avoid tedious purification procedure, sercretory expression of ADI in E. coli and Pichia pastoris was studied.P. plecoglossicida Arginine deiminase (ADI) encoding gene arcA was optimized according codon usage in P. pastoris. The optimized gene yh-ADI was inserted into vector pPIC9K. The P. pastoris GS115/pPIC9K-yh-ADI recombinants were screened in G418 plates after incubation in 96-well microtiter plates. In shake flask cultivation, methanol was added to a concentration of 1% for 72 h of induction, the ADI activity of P. pastoris GS115/pPIC9K-yh-ADI were 29.0 mU/mL broth.The secretory expression of ADI was also investigated in E. coli. The arginine deiminase (ADI) encoding gene of P. plecoglossicida CGMCC2039 was cloned into secreted expression vector pBAD/gⅢB under araBAD promoter (pBAD), and the resulted recombinant plasmid pBAD-ADI was transformed into E. coli TOP10F'. The effect of induction conditions on ADI expression was investigated, including L-arabinose concentration, induction temperature, and induction time. The optimum induction conditions were determined to be 0.002% L-arabinose, 25℃and 5 h, and the ADI activity of whole cells was 68 mU/mL broth. The recombinant ADI in periplasmic space was released by osmotic shock, the ADI activity in periplasmic and cellular fraction were 53 mU/mL broth and 34 mU/mL broth, respectively. SDS-PAGE analysis showed that the molecular mass of recombinant ADI was about 46 kDa.In this study, ADI from P. plecoglossicida was secretory expressed in E. coli and Pichia pastoris with the same biological activity as native enzymes. This work, for the first time, reports the expression of prokaryotic ADI in eukaryotic expression system. This study provides helpful experience for the further expression of ADI as fusion protein in Pichia pastoris, and also promote its application as an anti-cancer drug.