title: Single-step purification of cyclotides using affinity chromatography.
abstract: Cyclotides are considered promising scaffolds for drug development owing to their inherent host defence activities and highly stable structure, defined by the cyclic cystine knot. These proteins are expressed as complex mixtures in plants. Although several methods have been developed for their isolation and analysis, purification of cyclotides is still a lengthy process. Here, we describe the use of affinity chromatography for the purification of cyclotides using polyclonal IgG antibodies raised in rabbits against cycloviolacin O2 and immobilized on NHS-activated Sepharose columns. Cycloviolacin O2 was used as a model substance to evaluate the chromatographic principle, first as a pure compound and then in combination with other cyclotides, that is, bracelet cyclotide cycloviolacin O19 and Möbius cyclotide kalata B1, and in a plant extract. We demonstrate that single-step purification of cyclotides by affinity chromatography is possible but cross reactivity may occur between homologue cyclotides of the bracelet subfamily.
authors.affiliation: Division of Pharmacognosy, Uppsala University, Biomedical Center, Uppsala, SE, 75123, Sweden.|Department of Medicinal Chemistry, Uppsala University, Biomedical Center, Box 574, Uppsala, SE, 75123, Sweden.|Pharmacy Discipline, Khulna University, Khulna, 9208, Bangladesh.
authors.forename: Shaikh Jamal
authors.initials: SJ
authors.lastname: Uddin
authors.affiliation: Division of Pharmacognosy, Uppsala University, Biomedical Center, Uppsala, SE, 75123, Sweden.|Department of Medicinal Chemistry, Uppsala University, Biomedical Center, Box 574, Uppsala, SE, 75123, Sweden.
authors.forename: Taj
authors.initials: T
authors.lastname: Muhammad
authors.affiliation: Division of Pharmacognosy, Uppsala University, Biomedical Center, Uppsala, SE, 75123, Sweden.|Department of Medicinal Chemistry, Uppsala University, Biomedical Center, Box 574, Uppsala, SE, 75123, Sweden.
authors.forename: Md
authors.initials: M
authors.lastname: Shafiullah
authors.affiliation: W. Szafer Institute of Botany, Polish Academy of Science, Cracow, 31-512, Poland.
authors.forename: Blazej
authors.initials: B
authors.lastname: Slazak
authors.affiliation: Pharmacy Discipline, Khulna University, Khulna, 9208, Bangladesh.
authors.forename: Razina
authors.initials: R
authors.lastname: Rouf
authors.affiliation: Division of Pharmacognosy, Uppsala University, Biomedical Center, Uppsala, SE, 75123, Sweden.|Department of Medicinal Chemistry, Uppsala University, Biomedical Center, Box 574, Uppsala, SE, 75123, Sweden.
authors.forename: Ulf
authors.initials: U
authors.lastname: Göransson
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000737:chemistry* / Q000276:immunology; D002846:Chromatography, Affinity / Q000379:methods*; D047169:Cyclotides / Q000737:chemistry* / Q000276:immunology / Q000302:isolation & purification*; D047168:Cystine Knot Motifs / Q000276:immunology; D011817:Rabbits
chemical_list: D000906:Antibodies; D047169:Cyclotides; C091930:kalata B1; C473355:cycloviolacin O2
keywords: affinity chromatography; cyclotides; cycloviolacin O2; immobilized IgG