Cloning, expression of the abrin-a A-chain in Escherichia coli and measurement of the biological activities in vitro.
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 IC(50) 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.
Qing
Liu-Ting
LT
College of Animal Sciences, Huazhong Agricultural University, Wuhan 430070, China. qingliuting@yahoo.com.cn
Qu
Xiao-Ling
XL
D000036:Abrin / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D006026:Glycoside Hydrolases / Q000378:metabolism; D008862:Microsomes, Liver / Q000378:metabolism; D009699:N-Glycosyl Hydrolases / Q000378:metabolism; D014176:Protein Biosynthesis; D011506:Proteins / Q000187:drug effects; D012339:RNA, Ribosomal, 28S / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D054788:Ribosome Inactivating Proteins
D018076:DNA, Complementary; D011506:Proteins; D012339:RNA, Ribosomal, 28S; D011994:Recombinant Proteins; D000036:Abrin; D006026:Glycoside Hydrolases; D009699:N-Glycosyl Hydrolases; D054788:Ribosome Inactivating Proteins