Construction, Expression, Purification And Modification Of MPEG Of Diphtheria Toxin-related Interleukin 2(DAB₃₈₉-IL2)

Immunotoxin DAB389-IL2 is a diphtheria toxin-based fusion protein which contain both full length human Interleukin 2(IL2) and the enzymatic domain and transmembrane domain of Diphthreia toxin(DT). DAB389-IL2 can specifically kill cells with IL2 receptors on their surfaces, and the mechanism is that once IL2 molecules bind to their receptors on cell surfaces. the immonotoxin will enter cells by endocytoxins, then, the transmembrane domain will translocate the enzymatic domain into the cytoplasm, and the later will inhibit the protein synthesis of the cell and kill the cell. DAB389-IL2 are widely used in the studies on targeted drugs, on the cell surface of some tumors the receptors of IL2 has been demonstrated beening at a very high level, thus, it is very useful on targeting therapy for tumor deseases. In this work, recombinant plasmid pET-28a/His6-DAB389-IL2 was constructed, and protein His6-DAB389-IL2 was expressed and purified. we took some researches on the mPEG chemical modification of the fusion protein His6-DAB389-IL2, and studyed the cytotoxicity of Mpeg-DAB389-IL2. we constructed recombinant plasmid pET-30a/His6-IL2. First, we got His6-IL2 gene by polymerase chain reaction(PCR) amplification procedures, then His6-IL2 was cloned into pcDNAII vector. After treatment of restrict enzymes (BamHI and NdeI), His6-IL2 was inserted into the corresponding site of the prokaryotic expression vector pET-30a, the correct vector was named as pET-30a/His6-IL2. We also construct expression vector pET-28a/His6-DAB389-IL2. Tthe DAB389-IL2 gene fragment was cut from plasmid pET-30a/DAB389-IL2, then it was inserted into the correspongding sites of the expression vector pET-28a. The gene of pET-28a contain his6 tag which can't be removed by NdeI, after expression the protein will take his6 tag, which will play important role in the purification of DAB389-IL2 by Ni2+-chelated affinity chromatography. The pET-30a/His6-IL2 and pET-28a/DAB389-IL2 plasmids were transformed into E.coli strain BL21, and the His6-IL2 and poly His-DAB389-IL2 protein were expressed successfully using 0.5mM IPTG to induce the transcription T7 promoters at 37℃for 5h.The molecular weight of the protein we got was about 17kD and 60kD, which is as expected. Western-blot analysis of His6 -DAB389-IL2 showed that the protein could react well with anti-DTA McAb, anti-poly-His McAb and anti-IL2 antibodies repectively, which indicated that the protein contained poly-His, IL2 and Diphtheria A fragment. The His6-DAB389-IL2 was purified by Ni2+ Sepharose chromatography. The pellets were ultrasonicated, and the inclusion bodies(IB) were denatured by 8M urea, and renatured on Ni2+ Sepharose column by enducing the urea concentration gradually for about 16 hours, then we got the renatured protein by washing the column with 0.5M imidazole on FPLC system. At last a purity about 97% can be achieved by Ni2+ Sepharose. We also studied the mPEG chemical modification of His6-DAB389-IL2, and the cytotoxicity of DAB389-IL2 after modification. A solution about 0.5 mg/mL of His6-DAB389-IL2 in 0.1M phosphate buffer, pH 5.0 was added to a vial containing 10 times amount of ALD-mPEG in the presence of 20mM sodium cyanoborohydride (NaCNBH3) in water. The reaction was stirred at 4°C for 24...