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Oxford University Press (OUP), Protein Engineering, Design & Selection, 7(10), p. 751-755

DOI: 10.1093/protein/10.7.751

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N-Terminal truncation mutagenesis of equinatoxin II, a pore-forming protein from the sea anemone Actinia equina

Journal article published in 1997 by G. Anderluh ORCID, J. Pungercar, I. Krizaj, B. Strukelj, F. Gubensek, P. Macek
This paper is made freely available by the publisher.
This paper is made freely available by the publisher.

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Abstract

The role of the N-terminal segment 1-33 of equinatoxin II, a 20 kDa pore-forming protein from the sea anemone Actinia equina, was studied by N-truncation mutagenesis. A part of this segment was classified as being amphiphilic and membrane seeking. Wild-type equinatoxin II and its mutants lacking 5, 10 and 33 amino acid residues, respectively, were produced in Escherichia coli using T7 RNA polymerase-based expression vector. Soluble recombinant proteins were isolated from bacterial lysates and assayed for their inhibition by sphingomyelin, binding to red blood cells and hemolytic activity. The N-terminal deletion of 33 amino acids resulted in an insoluble protein, while mutants lacking 5 and 10 residues expressed increased relative avidity for sphingomyelin and red blood cell membranes. Their specific hemolytic activity was decreased, however, with increasing truncation. The results suggest that the N-terminus, which has been found to be conserved in sea anemone pore-forming toxins, contributes to the solubility of the equinatoxin II, but it is not essential for binding to lipid membranes. It is very likely that the N-terminus play a role in the formation of functional pores.