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- Title
Phenotypic selection and characterization of randomly produced non-haemolytic mutants of the toxic sea anemone protein sticholysin II
- Authors
Alegre-Cebollada, Jorge; Lacadena, Valle; Oñaderra, Mercedes; Mancheño, José M.; Gavilanes, José G.; del Pozo, Álvaro Martínez
- Abstract
A rapid screening method for haemolytic activity, using blood agar plates, has been developed to analyze randomly produced mutant variants of the pore-forming protein sticholysin II (Stn II). Those exhibiting a reduced activity were selected and the DNA corresponding to each Stn II variant sequenced. Once the mutation produced was determined, protein variants were isolated and characterized in terms of structure (circular dichroism spectra and thermal stability) and haemolytic activity. Three single mutation protein variants, at residues K19, F106 and Y111, showed a significantly decreased haemolytic activity while their thermostability was identical to that of the wild-type protein. Considering the obtained data and based on the three-dimensional structure of the protein, the role of these residues on the mechanism of haemolysis has been analyzed.
- Subjects
PHENOTYPES; GENETIC mutation; SEA anemones; ZOANTHARIA
- Publication
FEBS Letters, 2004, Vol 575, Issue 1-3, p14
- ISSN
0014-5793
- Publication type
Article
- DOI
10.1016/j.febslet.2004.08.031