Phenotypic selection and characterization of randomly produced non-haemolytic mutants of the toxic sea anemone protein sticholysin II

被引：34

作者：

Alegre-Cebollada, J

Lacadena, V

Oñaderra, M

Mancheño, JM

Gavilanes, JG

del Pozo, LM ^{[1
]}

机构：

[1] Univ Complutense, Fac Quim, Dept Bioquim & Biol Mol, E-28040 Madrid, Spain

[2] CSIC, Inst Quim Fis Rocasolano, Grp Cristalog, Madrid 28006, Spain

来源：

FEBS LETTERS | 2004年 / 575卷 / 1-3期

关键词：

actinoporin; equinatoxin; haemolysis screening; random mutation; protein structure;

D O I：

10.1016/j.febslet.2004.08.031

中图分类号：

Q5 [生物化学]; Q7 [分子生物学];

学科分类号：

071010 ; 081704 ;

摘要：

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. (C) 2004 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

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页码：14 / 18

页数：5

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