Basic Research Of A New Generation Pegylated ADI

Objective:Normal cells do not require arginine for growth,since they can synthesize arginine from citrulline with a two-step process.In contrast,it has been reported that primary hepatocellular carcinoma cells requires arginine for growth,so they are auxotrophic for arginine.Arginine deiminase(ADI),an enzyme that degrades arginine,may be utilized as a treatment means.This research tries to develop a new generation drug for hepatomas:Pegylated ADI(PEG-ADI).Methods:The ADI gene was produced by denovo synthesis,while Escherichia coli preference codons were introduced.The synthesized gene fragment was inserted to pET3a and the recombinant plasmid contained ADI gene(pET3a-ADI) was transformed to Escherichia coli BL21DE3(pLyss).Escherichia coli BL21DE3(pLyss) harboring ADI was induced by incubation with IPTG for 4h,and the cells were harvested by centrifugation.The cell pellets were resuspended in PB and disrupted by high pressure.The inclusion bodies were solubilized with 8M Gu-HCl and renaturation was performed under refolding conditions.The resulting completely refolded rADI was purified using sequential Q-Sepharose anion exchange and Phenyl HP hydrophobic interaction and Arginine Sepharose 4B.ADI enzyme activity was determined by the production of L-citrulline from L-arginine.The PEG was covalently binded with rADI by a new generation PEG modification technology.The resulting PEG-rADI was purified using Resource PHE hydrophobic interaction.The number of PEG molecules attached per ADI molecule was determined by Fe(SCN)₃.The mensuration of PEG-rADI enzyme activity was the same as the one of ADI.The in vivo and in vitro animal pharmacodynamics and Pharmacokinetics experiments were performed by in vitro hepatomas inhibition, pharmacodynamic effects on H22 implant tumor,and immunogenicity assessment of normal rice.Result:An Escherichia coli expression vector harboring rADI(pET3a-ADI) was constructed successfully.By improving fermentation conditions,improving the expression of object protein,and optimizing refolding conditions of rADI inclusion bodies,the protein recovery rate was promoted up to 40%.The specify activities of purified rADI were above 40IU/mg.And the new generation Pegylated rADI significantly decreased immunogenicity comparing with unmodified rADI.The in vivo and in vitro animal pharmacodynamics and Pharmacokinetics experiments proved:The effect of tumor inhibition of this new generation Pegylated rADI was equal to PEG-ADI20 of Phoenix Pharmacologics Inc.Conclusions:A new generation pegylated ADI has been successfully constructed,and the effect of tumor inhibition of this PEG-rADI has been confirmed.