Identification of a novel cytotoxic protein, Cry45Aa, from Bacillus thuringiensis A1470 and its selective cytotoxic activity against various mammalian cell lines.
Parasporal inclusion proteins produced by Bacillus thuringiensis strain A1470 exhibit strong cytotoxicity against human leukemic T cells when activated by protease treatment. One of the cytotoxic proteins was separated by anion exchange chromatography and gel filtration chromatography and designated Cry45Aa. Its gene was then expressed in recombinant Escherichia coli, in which the Cry45Aa precursor was accumulated in an inclusion body. It was solubilized in sodium carbonate buffer and processed with proteinase K, and cytotoxic activities of the protein against various mammalian cell lines were evaluated using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H tetrazolium bromide assay. The protein exhibited high cytotoxic activity against CACO-2, Sawano, MOLT-4, TCS, and HL60 cells and moderate activity against U-937 DE-4, PC12, and HepG2 cells. On the other hand, the EC50 values against Jurkat, K562, HeLa, A549, Vero, COS-7, NIH3T3, CHO, and four normal tissue cells (human primary hepatocyte cells, UtSMC, MRC-5, and normal T cells) were >2 microg/mL.
Okumura
Shiro
S
Fukuoka Industrial Technology Center, 1465-5 Aikawa, Kurume, Fukuoka 839-0861, Japan. sokumura@fitc.pref.fukuoka.jp
Saitoh
Hiroyuki
H
Ishikawa
Tomoyuki
T
Wasano
Naoya
N
Yamashita
Satoko
S
Kusumoto
Ken-Ichi
K
Akao
Tetsuyuki
T
Mizuki
Eiichi
E
Ohba
Michio
M
Inouye
Kuniyo
K
D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology*; D001413:Bacillus thuringiensis / Q000737:chemistry*; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology*; D016923:Cell Death / Q000187:drug effects; D002460:Cell Line; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D011994:Recombinant Proteins / Q000494:pharmacology
D000970:Antineoplastic Agents; D001426:Bacterial Proteins; D011994:Recombinant Proteins