[High level expression, purification and cytotoxicity of IL-10(18-57)-PE40].
The objective of the experiment is to explore the purification and production of immunotoxin. The chimeric toxin, which is composed of 40 peptides of interleukin 10 (from amino acids 18 to 57) fused to a mutant form of Pseudomonas extoxin (PE) devoid of its native cell recognition domain. Two kinds of prokaryotic expression vector containing the chimeric toxin IL-10(18-57)-PE40 were constructed respectively. After induction of IPTG for 3 hours, IL-10(18-57)-PE40 was expressed highly in cytoplasmic fraction in Rosettablue(DE3), and was directed to periplasmic space as soluble form in E. coli BL21(DE3)pLysS . Western -blotting showed that the expressed protein could react with the specific rabbit sera against LHRH-PE40. With the application of salting out of (NH4)2SO4, hydrophobic interaction chromatography, Cu-affinity chromatography and anion exchange chromatography, the purity of IL-10(18-57)-PE40 was about 96%. The cytotoxicity assay, Cell-ELISA and fluorescent antibody test support the hypothesis that IL-10(18-57) based ligand-mediated cytotoxicity can serve to target cytotoxic agents in vitro.
Peng
Qi-Sheng
QS
College of Veterinary, Jilin University, Changchun 130062, China. pengqisheng@sina.com
Li
Yue-Hong
YH
Zhu
Ping
P
D036002:ADP Ribose Transferases / Q000096:biosynthesis* / Q000235:genetics; D001427:Bacterial Toxins / Q000096:biosynthesis* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005098:Exotoxins / Q000096:biosynthesis* / Q000235:genetics; D005822:Genetic Vectors; D006801:Humans; D000922:Immunotoxins / Q000235:genetics / Q000302:isolation & purification*; D016753:Interleukin-10 / Q000096:biosynthesis* / Q000235:genetics; D011550:Pseudomonas aeruginosa / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D037521:Virulence Factors / Q000096:biosynthesis* / Q000235:genetics; D000097668:Pseudomonas aeruginosa Exotoxin A
D036002:ADP Ribose Transferases; D001427:Bacterial Toxins; D005098:Exotoxins; D000922:Immunotoxins; D016753:Interleukin-10; D011993:Recombinant Fusion Proteins; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A; C508609:IL10 protein, human