Cloning,
Expression of the Abrin-a A-chain in Escherichia coli and Measurement of
the Biological Activities in vitro
QING Liu-Ting*, QU Xiao-Ling1ª¤
( College of Animal Sciences, Huazhong Agricultural University, Wuhan 430070,
China; 1Institute of Virology, College of Life Sciences,
Wuhan University, Wuhan 430072, China )
Abstract The coding
sequence of abrin-a A-chain (ABRaA) gene was obtained by RT-PCR and cloned into
the expression vector pET28b. The mature ABRaA has been highly expressed in the
cytoplasm of Escherichia coli by 1 mmol/L IPTG induction, and the yield
of the soluble recombinant protein was 4 mg/L of induced culture. The
recombinant ABRaA was purified to be homogeneity. The biological activities of
expressed ABRaA were demonstrated in vitro. It strongly inhibited the
protein biosynthesis of rabbit reticulocyte lysates, with an IC50 of
0.08 nmol/L. It also depurinated 28 S rRNA through cleaving at the A4324 site
in rat liver ribosomes by its N-glycosidase activity. These data
suggested that the recombinant ABRaA could be used for the preparation of
immunotoxins as a potential cancer chemotherapeutic agent.
Key words abrin-a A-chain (ABRaA);
ribosome-inactivating protein (RIP); cloning and expression; inhibition of
protein biosynthesis; N-glycosidase activity
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