Studies On Soluble Expression Optimization And Purification Of Recombinant Target Toxin IL6_（T23）-PE38KDEL

The recombinant immunotoxin has become one of the highly regarded tumortherapies. Compared with conventional methods, it has the advantage of betterspecificity, strong and fast efficiency, less dose and side effects. With thedevelopment of genetic engineering and gene-recombinant technologies, scientistshave made remarkable progress in expressing recombinant immunotoxin in bothprokaryotic and eukaryotic systems, which is represented by E. coli and Pichiapastoris, respectively. E.coli has since been the first choice due to its thorough geneticbackground, fast growth rate, high expression level and easy for fermentation.However, one main problem at home and abroad is the formation of inclusion bodiescontaining the target proteins in E. coli.This article is aimed to the research of eukaryotic expression of the targetedtoxin IL6_（T23）-PE38KDEL in Pichia pastoris strain SMD1168by vector pGAPZαA.However, at first the effect of recombinant immune toxin was no ideal because nobiological viability could be detected in cell activity test, probably because the vectorand host does not match. With the development of industrial production ofrecombinant immunotoxin, more attention was scattered to high density fermentation,which aims to raise production with relatively lower cost. The main factors that affectthe production include: components of culture medium, dose and kind of the inducingagent and the growth environment of the strain. Previously, we found thatIL6_（T23）-PE38KDEL expressed in prokaryotic system was maily in the form ofinclusion body, which complicated further purification and refolding.This study compared the difference of IPTG and lactose induction, anddemonstrated the feasibility of auto-inducible expression of IL6_（T23）-PE38KDEL forthe first time. And auto-induction medium components and recombinant bacteriaculture conditions were optimized by single factor experiments and orthogonal designmethods. The components of the medium include:2%peptone,1.5%yeast extract,25 mM Na2HPO4,25mM KH2PO4,25mM （NH4）2SO4,0.05%glucose,1%mannitol,1.0%lactose,15mM sodium malate,0.5mM MgSO4. Incubate2%recombinant Ecoli into a250mL flask containg40mL cluture medium,28℃,25h.IL6_（T23）-PE38KDEL is20.4%in total soluble protein, while the cell density is21.36and the plasmid stability is as high as98%. The experiment laid the foundation forlarge scale fermentation of recombinant immunotoxin.We optimized the method of IL6_（T23）-PE38KDEL expression and purification.Firstly, the recombinant toxin was cushy purified by20%to40%two-stepammonium sulfate precipitation, and then followed by hydrophobic interactionchromatography and ion exchange chromatography. Finally the purity of solubleIL6_（T23）-PE38KDEL is88%, which showed great lethality to myeloma cells SP2/0.