Cloning, expression and cytotoxicity of Pseudomonas aeruginosa exotoxin PE38KDEL with high content of GC
Objective To obtain a modified exotoxin PE38KDEL with low molecular mass and high activity. Methods PCR and enzyme digestion technology were used to construct the recombinant exotoxin PE38KDEL. The resulting gene encoding PE38KDEL was expressed in E.coli and the expression product was identified by Western blot assay. In order to test the biological activity of PE38KDEL, a fusion protein was constructed by genetically fusing PE38KDEL to interleukin-4 mutant cpIL4(13D) and was expressed in E.coli. After purificatioin, the fusion protein was tested for its cytotoxicity to melanoma cell line LiBr that expresses IL-4R using MTT technology. Results The gene of PE38KDEL was successfully constructed and expressed in E.coli BL21(DE3). Fusion protein cpIL4(13D)-PE38KDEL was constructed and expressed in E.coli AD494(DE3). The purity of the fusion protein was above 95%. cpIL4(13D)-PE38KDEL showed significant cytotoxicity to IL4R-bearing tumor cell line LiBr. Conclusion The construction of PE38KDEL laid a solid foundation for the development of immunotoxins for potential tumor therapy.