Expression and purification of the anticomplementary peptide Sh-CRIT-ed1 (formerly Sh-TOR-ed1) as a tetramultimer in Escherichia coli.

Many complement inhibitors found in plants and other organisms have been recognized as an antiinflammatory drug. Sh-CRIT-ed1 is a complement inhibitory peptide, present on the Schistosoma parasite surface. In the present study, we expressed chemically synthesized oligonucleotides encoding Sh-CRIT-ed1 with an additional hexahistidine codon at the C-terminus and purified in Escherichia coli BL21. The cloned gene, which was multimerized four times in pBlue-script II KS (+) at the isoschizomer sites (BamHI, BglII), was named Sh4, and expressed in E. coli BL21 harboring pGEX-KG. The fusion protein (GST-Sh4) was purified with high yield successively by affinity chromatographies of glutathione-Sepharose 4B and Ni-NTA-agarose. Recombinant Sh-CRIT-ed1 was obtained readily by thrombin digestion and CNBr cleavage of GST-Sh4, and the yield was 9.03 mg from 1-liter culture of E. coli BL21 harboring pGEX-Sh4. The recombinant Sh-CRIT-ed1 showed strong anticomplementary activity (IC(50) = 6.02 microM) by complement haemolysis assay. Copyright 2002 Elsevier Science (USA)