Preparation and characterization of fusion protein truncated Pseudomonas Exotoxin A (PE38KDEL) in Escherichia coli.
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-Ni(2+) 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 Hut 102 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 Hut 102 and hepatocellular cell lines. The results suggest that PE38KDEL would be useful for the preparation of more potent immunotoxins.
Song
Shuichuan
S
International Cancer Institute, The Second Military Medical University, 800 Xiang Yin Road, Shanghai 200433, PR China. shuichuansong@yahoo.com
Xue
Jingya
J
Fan
Kexing
K
Kou
Geng
G
Zhou
Qian
Q
Wang
Hao
H
Guo
Yajun
Y
D036002:ADP Ribose Transferases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D000595:Amino Acid Sequence / Q000235:genetics*; D000818:Animals; D001427:Bacterial Toxins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D002460:Cell Line; D002846:Chromatography, Affinity / Q000379:methods; D004926:Escherichia coli / Q000235:genetics; D005098:Exotoxins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D015870:Gene Expression / Q000235:genetics; D006801:Humans; D000922:Immunotoxins / Q000737:chemistry / Q000302:isolation & purification; D051379:Mice; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D017384:Sequence Deletion / Q000235:genetics*; D037521:Virulence Factors / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D000097668:Pseudomonas aeruginosa Exotoxin A
D036002:ADP Ribose Transferases; D001427:Bacterial Toxins; D005098:Exotoxins; D000922:Immunotoxins; D011993:Recombinant Fusion Proteins; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A