Preparation And Cytotoxicity Of Immunotoxin (IL-10_23-57-PE40)

Interleukin-10(IL-10) was first described as cytokine synthesis inhibitory factor(CSIF),which open reading frames(ORF) encode matured proteins of 160 amino acids.IL-10 is a multifunctional cytokine with diverse effects on most hemopoietic celltypes.The IL-10 receptor is composed of at least two subunits (IL-10Rα,IL-10β) thatare members of the interferon receptor(INFR) family,the ligand-binding subunit( IL-10Rα) binds IL-10 with high affinity(Kd～35-200pM),on the other hand, IL-10βcontibuteslittle to IL-10-binding affinity.By flow cytometry, IL-10Rαmainly expressed inlym-phoma cells such as mono-macrophage (200-300 receptors per cell),there appears tobe at most only a few receptors on nonlymphoid cells.Therefore, from the point ofcon-structing optimal immunotoxin(IT),IL-10 is suitly used to construt the targetingagents of IT.However,IL-10 is expressed in the form of inclusion body in E.coli,theIL-10-PE40 is surely inclusion body in E.coli if PE40 was recombined with IL-10.Inorder to solve the problem and apply a new method to construct targeting moiety,referingto the mapping of the IL-10/ IL-10Rαcombing site,we design the mimics peptide ofIL-10 as ligant instead of IL-10:IL-1023-57,IL-1018-57.Pseudomonas exotoxin A is a single chain bacterial toxin, which is composed ofthree structural domains. Each domain has each distinct function. Domain I is composedof domain Ia (amino acids l-252) and Ib (amino acids 365-399). Domain Ia is responsiblefor cell binding; however, the function of domain Ib remains obscure.Domain II (aminoacids 253-364) plays an essential role in the translocation of PE when it across the cellmembrane and into the cytosol.Domain III(amino acids 400-613) with ADP-ribosyltransferase activity can inactivate elongation factor 2 , and cause cell death.Derivatives ofnative Pseudomonas exotoxin have been developed and named PE40(253-613aa), Todevelop a specific cytotoxic agent ,chimeric toxin was made by fusingIL-1023-57,IL-1018-57with derivative PE40: IL-1023-57-PE40, IL-1018-57-PE40,respectively. By choosing optimal codons of E.coli,the IL-1023-57,IL-1018-57 gene fragmentwasam-plified by overlapping PCR . The PCR products were digested by NdeⅠandNcoⅠ,and then inserted into plasmid vector pET28a-PE40,respectively.Secondly,the newplas-mid pET28a-IL-1023-57-Pe40, pET28a-IL-1018-57-Pe40 were digested by NcoI andEcoRI,and then inserted into plasmid vector pET20b,respectivly.By sequencing the aboveco-nsturcting vector, pET28a-IL-1023-57-Pe40, pET28a-IL-1018-57-Pe40,pET20b-IL-1023-57-Pe40, pET20b-IL-1018-57-Pe40 were constructed correctly. The recombinant plasmid pET28a-IL-1023-57-Pe40 and pET20b-IL-1023-57-Pe40were transformed into E.coli BL21(DE3),BL21(DE3)pLysS,Rosetta(DE3)BL21,ER2566 and E. coli K12 TB1for proposed protein expression,respectively.Underinduc-tion of IPTG the recombinant protein IL-1023-57-Pe40 was expressed as a solubleprotein and was up to 15% of the total protein in Escherchia coli RosettaBL21(DE3),target pro-tein IL-1023-57-Pe40 directed to periplasmic space only in E.coliBL21(DE3)pLysS and was up to 10% of the total protein.Western blot analysis showedthat the fusion protein may react specifically with anti-PE40 antibody. The recombinant plasmid pET28a-IL-1018-57-Pe40 and pET20b-IL-1018-57-Pe40were transformed into E.coli BL21(DE3),BL21(DE3)pLysS,Rosetta(DE3)BL21,for proposed protein expression,respectively.Under induction of IPTG the recombinantprotein IL-1023-57-Pe40 was expressed as a soluble protein and was up to 20% of the totalprotein in Escherchia coli Rosetta BL21(DE3),target protein IL-1023-57-Pe40 didn'tdirected to periplasmic space in the above E.coli.Western blot analysis showed that thefusion protein may react specifically with anti-PE40 antibody. By rationale for plasmid stability test in E.coli BL21(DE3),Rosetta(DE3)BL21,ER2566 and E. coli K12 TB1, pET20b-IL-1023-57-Pe40 and pET20b-IL-1018-57-Pe40were only unstable target plasmids in BL21 ( DE3 ) ,that maybe explain whyIL-1023-57-Pe40 and IL-1018-57-Pe40 didn't directed to periplasmic space in BL21(DE3).However, why the target protein didn't export to periplasm of the other hoststrains ?Firstly,the signal peptide of plasmid is necessary,but not sufficient to export toperiplasm; secondly, translocation across the cell membrane of E.col also depend on themature domain of the target protein which is recognized by SecB,the major charperon ofexport;Thirdly,the first 14 to 18 residues of the mature sequence have the highestdeviation between the observed an expected net charge distibution,moreover,almost allseqences of the secreted protien have either neutral or negative net charge in thisregion;fourthly, the hydrophobicity of the first 20 residues of target protein plays anessential role in the translocation across of membrane of E.col;finally,the specificity ofhost strains was associated to lead recombined protein out of the cytoplasm. The designed fusion protein IL-1023-57-Pe40 and IL-1018-57-Pe40 were analyzedusing DNAStar in some molecular biology characteristics. It was predicted thathydrophobi-city of IL-1023-57-Pe40 and IL-1018-57-Pe40 were almost same as that ofPE40.The isoelectric point of IL-1023-57-Pe40 and IL-1018-57-Pe40 was 4.7,4.8respectively.There-fore,we design hydrophobic interaction chromatograrhy(HIC) and ionexchange chroma-tography(IE) as main method of purification for IL-1023-57-Pe40 andIL-1018-57-Pe40. High cell density fermentation of the recombinant E.coli strain was studied,foroptimizing the cell density and protein production. the expression reached at the top level3 hours after induction with 1mmol/L IPTG concentration. E.coli cell density could reach30g/L and the expression remains at high level. Cells was collected by centrifugation andlysed by sonication. Target Protein IL-1023-57-Pe40 was purified by Octyl Sepharose ,DEAE Sepharose and Cu affinity chromatography,respectively. With the abovepurification, the purity of IL-1023-57-Pe40 was about 90%. Target Protein IL-1018-57-Pe40precipitated by 35% ammonium sulfate and then purified by Phenol Sepharose, ButelSephrose, Cu affinity and Q Sephrose chromatography,respectively.With the above purification, the purity of IL-1018-57-Pe40 was about 95%.