Modification,Preparation And Characterization Of Human Soluble Tumor Necrosis Factor-related Apoptosis Inducing Ligand

Tumor necrosis factor-related apoptosis-inducing ligand(TRAIL)is a characterized member of the tumor necrosis factor(TNF)superfamily,which is expressed as a type 2 transmembrane protein containing 281 amino acids.Its extracellular region(114-281 aa)can be digested from the cell surface to form a soluble molecular(sTRAIL)with biological activity.Recently,sTRAIL has attracted considerable attention thanks to its selective anti-tumor effects in vitro and in vivo.However,the traditional isolation of sTRAIL from animal tissue or cell culture media is difficult to meet the increasing applications.Therefore,a more effective and economic preparation process is urgent needed to facilitate further research and development of sTRAIL-based biologicals.Previously,an increasing number of research have reveal that the recombinant human soluble TRAIL(rhsTRAIL)without glycosylation also maintained the anti-tumor activity of entire molecular.The prokaryotic bacterial such as E.coli is considered as the most cost-effective system for rhsTRAIL expression and preparation.Unfortunately,refolding of inclusion body is a tedious and difficult process,which limits the effective preparation of many kinds of therapeutic proteins.In this work,to improve the soluble expression of rhsTRAIL in E.coli,a conventional site-directed mutation was used to replace the amino acid residues at the N-terminus of sTRAIL protein.Our research work is detailed as followed:1.Construction,selection and characterization of sTRAIL mutantsFirstly,a novel fusion gene,composed of the encoding sequences of 5'-terminus polyhistidine-tag(His),interval cleavage-site of tobacco etch virus protease(TEV)and 3'-terminus wild sTRAIL,was prepared by using 2-rounds add-PCR amplification.After digestion with Nco? and Hind?,the purified fusion fragment was cloned into the pET-28a to construct expression vector pET28a/His-sTRAIL.Secondly,the above-mentioned recombinant plasmid(pET28a/His-sTRAIL)was used as a template to build 4 plasmid mutants by site directed mutagenesis,in which residues in the wild sTRAIL was respectively substituted for alanine at 4 different selected positions.Further expression analysis discovered that the substitution for alanine(Ala)at position B significantly improve soluble expression of sTRAIL in E.coli.Thirdly,the alanine at position B was further substituted for other 4 different type of amino acids,including Aspartic acid(Asp),Serine(Ser),Glycine(Gly)and Cysteine(Cys).As a result,a mutant sTRAIL(msTRAIL)with alanine substitution at position B was screened out as the robust mutagenesis for further research.2.Preparation and identification of msTRAILAccording to the properties of msTRAIL,a series of protein manipulates,including IPTG inducing at low temperature,soluble protein extraction,ammonium sulfate precipitation,Ni affinity chromatography and TEV digestion,were combined into an efficient and economic process to produce this novel sTRAIL.Moreover,the molecular weight,purity,protein concentration,antigenicity,N-terminal sequence and secondary structure of msTRAIL was further identified by using SDS-PAGE,BCA assay,Western-blot,Edman degradation and circular dischroism chromatography respectively.3.Characterization of the anti-tumor effect of msTRAILTo investigate the anti-tumor effect of our novel msTRAIL,CCK8 testing,Annexin V/PI double staining and Caspase3 spectrophotometric assays was respectively used to evaluate its cytotoxicity,apoptosis induction,as well as apoptotic enzyme activation.Fortunately,all of these evaluation suggest that our screen out novel sTRAIL maintains the anti-tumor activities of native molecular.Meanwhile,another animal vaccination also discovered that this single mutation in msTRAIL does not enhance anti-sTRAIL immunogenicity which is undesirable in clinical application of therapeutic biologicals.In conclusion,sTRAIL,a biological candidates against cancer,was modified by using site-directed mutangensis in this work.We successfully discovered that the residues in the N-terminus of sTRAIL could significant influence its soluble expression in E.coli.A robust mutant sTRAIL(msTRAIL)was fortunately screened out from 8 sTRAIL mutants,which represent significant improving soluble expression compared with the wild sTRAIL.Consequently,more than 31.9 mg purified msTRAIL with 95%purity could easily and rapidly produced from 1 liter bacterial cultures by using our build cost-effective process.More importantly,our site-directed mutangenesis in sTRAIL not only maintain the native anti-tumor effect of wild molecular,but also avoid strength harmful anti-TRAIL immune response in vivo.We hope our novel msTRAIL could make a positive contribute to further research and development of anti-tumor biotherapeutics based on sTRAIL.