Cloning And Expression Of Cytotoxin Ⅲ From Naja Naja Atra And Its Mechanism On Inhibition Of Tumor Cells

The major components from snake venom are pharmaceutically complex of proteins and peptides which exert different biological activities.Some proteins induce death as neurotoxin,cardiotoxin or organic necrosis,while there are also some low toxic components which induce different pharmaceutical effects.These toxins affect normal physiological process through attack the specific sites on target cells. Cytotoxins from Naja naja atra are basic polypeptides which bind to susceptible cells on proteins or membrane phospholipids.In recent years,purified cobra snake venom components were widely applied on different solid tumor and leukemia study.In this article,we summarized the cloning,expression and purification of recombinant cytotoxinâ…¢(CTXâ…¢),and the application as chemotherapeutic drug on anti-tumor therapy.There were three major parts in this dissertation.1.The physiological characteristics of native CTXâ…¢from cobra(Naja naja atra) were obtained by physical-chemical analysis,the CTXâ…¢had a purity of more than 95%by RP-HPLC and TOF-MASS.The N-terminal sequence of CTXâ…¢was determined by Ediman degradation method.The gene of CTXâ…¢was obtained by RT-PCR method and was cloned into two prokaryotic expression vectors and eukaryotic yeast Pichia pastoris pPIC9K vector.The recombinant protein from pPEY30a was expressed as insoluble inclusion bodies.CTXâ…¢gene,fused with enterokinase in E.coli His-patch Thioredoxin expression system,was expressed in soluble form and released by osmotic-shock treatment.The recombinant CTXâ…¢was released from fusion protein by enterokinase lysis.The production and purification process were performed at lab scale.About 7 mg recombinant CTXâ…¢was obtained from per liter of bacteria culture with overall recovery of 37%.While the yield of recombinant CTXâ…¢from pichia pastoris pPIC9K was about 9.5 mg from per liter medium.Using straightforward one-step chromatography procedure,the rCTXâ…¢, with three additional amino acids(GYT) at the N-terminal site,was purified to a purity of more than 95%and recovery yield of 65%.The purified rCTXâ…¢was further characterized by hemolytic activity and cytotoxic assay compared with natural CTXâ…¢with HD₅₀ 6.53μg/ml and7.4μg/ml,respectively,and IC₅₀ 3.56μg/ml and 2.63μg/ml,respectively,suggested that the activity of rCTXâ…¢was similar to the nCTXâ…¢and the rCTXâ…¢have the right conformation.The three additional amino acids(GYT) at the N termini of the rCTXâ…¢did not affect the formation of disulfide bonds and cytotoxicity of the protein.The Eukaryotic vector was selected as the ideal expression form for CTXâ…¢.2.Several methods including Annexin V/PI staining assay,Caspase activeity test and DNA fragmentation analysis,were used to evaluate the apoptosis of K562 cells induced by CTXâ…¢.Data showed that CTXâ…¢inhibited proliferation of human leukemia K562 cells by G2/M phase arresting and apoptosis which was associated with the activation of caspase-8 and cytochrome c release,inducing the subsequent truncation of Bid.Western blotting detected the phosphorylatin of p38 MAPK and JNK when K562 cells were exposed to CTXâ…¢.CTXâ…¢mediated apoptosis and phosphorylation of K562 cells were reduced by treatment with the MAPK-specific inhibitors indicated the activation of both JNK and p38 signaling pathway.A novel in vivo system-chicken chorioallantoic membrane-was developed for rapid assessment of leukemia k562 cell growth and treatment by chemotherapeutic CTXâ…¢.Large doses of CTXâ…¢did not infect the growth of embryo noted that CTXâ…¢has little toxicity toward organism.Daily administration of CTXâ…¢for two days to chicken chorioallantoic membrane(CAM) bearing tumours derived from the CAM at E10 administration of K562 cells resulted in inhibition of the tumours in vivo.Importantly, this in vivo inhibition was also associated with caspase-8 activation and cytochrome c release.Taken the formation of K562 cells into account,a hypothesis thus consider that CTXâ…¢competitively binding to ATP and block off the aberrant tyrosine kinase consecutive binding with ATP.These may thus explain the high efficacy of clinical treatment of CTXâ…¢.3.CTXâ…¢on solid tumor therapeutic effects were next analyzed.In this experiment, we investigated whether CTXâ…¢affects cell growth and cell cycle progression of hepatocellular carcinoma cells.We found that the proliferation of HepG2 cell was inhibited by CTXâ…¢,to some extent,in a time- and dose-dependent manner(IC50 2.58 mg/ml at 24 h) but little effects on Bel-1402 cells.DAPI staining,flow cytometric analysis and annexin V labeling also demonstrated that CTXâ…¢increased the percentage of apoptotic cells being associated with cell cycle arrest at S-phase. The apoptotic processs was associated with factor associated suicide and cytochrome c releasing.Semi-quantitative reverse transcription-polymerase chain reaction and western blot revealed that cyclin D1,cyclin A and cyclin E,which involved in cell apoptosis and cell cycle progression,were down regulated both at transcription and transcription levels.CTXâ…¢-induced caspase-8,-9 and caspase-3 activation, generation of truncated Bid,releaseing of cytochrome c and the change of Bcl-2/Bax ratio on protein and mRNA levels.In vivo tests detected that CTXâ…¢would cause sudden death when i.p.injected with LD50 936.7±38μg/kg.Tumor growth inhibition was most evident in mice treated with CTXâ…¢at 2 mg/kg/d,where an approximately 39.4%reduction in tumor size was observed,in contrast with mice treated with saline. No obvious toxicity,as judged by parallel monitoring of body weight and tissue sections of heart,intestine,liver and lung,was observed in CTXâ…¢-treated mice.In conclusion,CTXâ…¢could effectively inhibit the proliferation of tumor cells (K562 cells,HepG2 cells) both in vitro and in vivo.Recombinant CTXâ…¢from pichia pastoris has the similar biological activity compared to native CTXâ…¢.More importantly,this protocol can be easily used for the production of the toxin at a larger scale with low cost.The approach outlined in this report will be suitable for the production of other recombinant proteins especially those of the 'three-finger' family.