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Fractionation protocol for the isolation of polypeptides from plant biomass
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Division of Pharmacognosy.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Division of Pharmacognosy.
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1998 (English)In: Journal of natural products (Print), ISSN 0163-3864, E-ISSN 1520-6025, Vol. 61, no 1, 77-81 p.Article in journal (Refereed) Published
Abstract [en]

A fractionation protocol for the isolation of a highly purified polypeptide fraction from plant biomass is described. The procedure dereplicates ubiquitous substance classes known to interfere with bioassays often used in natural product-based drug discovery programs. The protocol involves pre-extraction with dichloromethane, extraction with ethanol (50%), removal of tannins with polyamide, removal of low-molecular-weight components with size-exclusion chromatography over Sephadex G-10, and final removal of salts and polysaccharides with solid-phase extraction using reversed-phase cartridges. The method has been applied to the aerial parts of Viola arvensis, resulting in the isolation of a peptide fraction that on further separation yielded a novel 29-residue macrocyclic polypeptide named varv peptide A, cyclo(-TCVGGTCNTPGCSCSWPVCTRNGLPVCGE-).

Place, publisher, year, edition, pages
1998. Vol. 61, no 1, 77-81 p.
National Category
Medical and Health Sciences
URN: urn:nbn:se:uu:diva-89791DOI: 10.1021/np970342rPubMedID: 9548831OAI: oai:DiVA.org:uu-89791DiVA: diva2:161545
Available from: 2002-04-05 Created: 2002-04-05 Last updated: 2012-01-24Bibliographically approved
In thesis
1. Macrocyclic polypeptides from plants
Open this publication in new window or tab >>Macrocyclic polypeptides from plants
2002 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The aim of this work was to explore the structural and functional diversity of polypeptides that are found in plants. Expanding knowledge of simililarities between plant use of these compound and animal use promises exceptional opportunities for finding, from plant research, new structures with biomedical and biotechnological potential.

A fractionation protocol was developed and applied to many plant species, providing fractions enriched in polypeptides, amenable to chemical and biological evaluation. From one species, the common field pansy (Viola arvensis), a 29-amino-acid residue polypeptide was isolated, named varv A, which revealed a remarkable macrocyclic structure (i.e., N- and C-termini are joined) stabilised by three knotted disulfides.

Varv A, together with an increasing number of homologous peptides, form the currently known peptide family of cyclotides. Their stable structure makes them an attractive scaffold for protein engineering. In addition, they display a wide range of biological activities (e.g., antimicrobial, cytotoxic, and insecticidal). As a part of this work, the cytotoxic effects of varv A and two other isolated cyclotides were evaluated in a human cell-line panel: all were active in the low µM range. Most likely, these effects involve pore formation through cell membranes.

Cyclotides were found to be common in the plant family Violaceae; with eleven cyclotides isolated and sequenced from V. arvensis, V. cotyledon, and Hybanthus parviflorus. For six members of the genus Viola, cyclotide expression profiles were examined by liquid chromatography-mass spectrometry (LC-MS): all expressed notably complex mixtures, with single species containing more than 50 cyclotides. These profiles reflect the evolution of the genus.

To assess these mixtures, a rational strategy for MS based amino acid sequencing of cyclotides was developed, circumventing inherent structural problems, such as low content of positively charged amino acids and the macrocyclic structure. This was achieved by aminoethylation of cysteines, which, following tryptic digestion, produced fragments of size and charge amenable to MS analysis. This method was also modified and used for mapping of disulfide bonds.

Methods for isolation and characterisation developed in this work may prove useful not only for further studies on macrocyclic polypeptides from plants, but also for other plant peptides and disulfide-rich peptides from animals.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2002. 59 p.
Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy, ISSN 0282-7484 ; 270
Pharmaceutical chemistry, Farmaceutisk kemi
National Category
Medicinal Chemistry
Research subject
urn:nbn:se:uu:diva-1956 (URN)91-554-5279-5 (ISBN)
Public defence
2002-04-26, BMC sal B22, Uppsala, 10:15
Available from: 2002-04-05 Created: 2002-04-05 Last updated: 2013-06-10Bibliographically approved

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