[Effective expression of ribonuclease-onconase in Escherichia coli and assaying its cytotoxic potential].
Onconase, a ribonuclease purified from Rana pipiens oocytes, has cytotoxic activity against several tumor cell lines in vitro. With the characteristic of containing four pairs of disulfide bonds internal and N terminal sequence attributing mostly to its biological functions, it is difficult to obtain the active Onconase from Escherichia coli expression system with normal strategy. Here, we fused the cDNA coding for Onconase in frame with the pelB signal sequence in pET22b(+) expression vector. Onconase can be effectively expressed in inclusion body without additional residues at N terminal under the proper inducing condition. After refolding and purifying process, we can get the recombined Onconase which has a similar ribonuclease activity as the one isolated from oocytes. The recombined Onconase has a pronounced effect on proliferation of Hut-78 cells (IC50=0.5 micromol/L).
Xu
Guo Feng
GF
School of life science, Zhejiang University, Hangzhou 310027.
Wang
Qing Cheng
QC
Gong
Xing Guo
XG
Fei
Jian
J
D000818:Animals; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008099:Liver / Q000378:metabolism; D051379:Mice; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D012313:RNA / Q000378:metabolism; D012260:Ribonucleases / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology
D011500:Protein Synthesis Inhibitors; D012313:RNA; D012260:Ribonucleases; C058703:ranpirnase