title: Expression and purification of the anticomplementary peptide Sh-CRIT-ed1 (formerly Sh-TOR-ed1) as a tetramultimer in Escherichia coli.
abstract: 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.
authors.affiliation: Graduate School of Biotechnology, Korea University, Seoul 136-701, Republic of Korea.
authors.forename: Kwang-Seok
authors.initials: KS
authors.lastname: Oh
authors.forename: Do-Kyun
authors.initials: DK
authors.lastname: Na
authors.forename: Mee-Hyang
authors.initials: MH
authors.lastname: Kweon
authors.forename: Ha-Chin
authors.initials: HC
authors.lastname: Sung
mesh_terms: D001483:Base Sequence; D003001:Cloning, Molecular; D003062:Codon; D004247:DNA / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000378:metabolism; D006461:Hemolysis; D006801:Humans; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000737:chemistry; D010455:Peptides / Q000737:chemistry*; D010957:Plasmids / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry
chemical_list: D003062:Codon; D009841:Oligonucleotides; D010455:Peptides; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D004247:DNA