Preparation and characterization of fusion protein truncated Pseudomonas Exotoxin A (PE38KDEL) in Escherichia coli

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• 作者：Shuichuan Song ; Jingya Xue ; Kexing Fan ; Geng Kou ; Qian Zhou ; Hao Wang and Yajun Guo
• 关键词：PE38KDEL ; Immunotoxin ; Affinity chromatography ; Luciferase
• 刊名：Protein Expression and Purification
• 出版年：2005
• 出版时间：2005
• 年：2005
• 卷：44
• 期：1
• 页码：52-57
• 全文大小：201 K

文摘

Pseudomonas Exotoxin A (PE) and truncated PE have been used to prepare immunotoxin with monoclonal antibodies. Truncated Pseudomonas Exotoxin A (PE38KDEL) was expressed with the pET-32a(+) vector in Escherichia coli under control of a T7 promoter. The recombinant protein was purified by His-Ni²⁺ metal affinity chromatography and gel filtration. The biological activity of PE38KDEL was evaluated by the inhibition assay of protein synthesis in rabbit reticulocyte lysate system, and the cytotoxicity was tested in Hut102 and hepatocellular cell lines by the MTS assay. PE38KDEL can significantly inhibit luciferase synthesis in cell-free protein synthesis assay and was slightly cytotoxic in the Hut102 and hepatocellular cell lines. The results suggest that PE38KDEL would be useful for the preparation of more potent immunotoxins.