Avtor/Urednik		Anderluh, Gregor; Pungerčar, Jože; Križaj, Igor; Štrukelj, Borut; Gubenšek, Franc; Maček, Peter
Naslov		N-terminal truncation mutagenesis of equinatoxin II, a pore-forming protein from the sea anemone Actinia equina
Tip		članek
Vir		Protein Eng
Vol. in št.		Letnik 10, št. 7
Leto izdaje		1997
Obseg		str. 751-5
Jezik		eng
Abstrakt		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.
Deskriptorji		CNIDARIAN VENOMS SEA ANEMONES MUTAGENESIS ESCHERICHIA COLI PROTEIN ENGINEERING AMINO ACID SEQUENCE BASE SEQUENCE DNA PRIMERS ERYTHROCYTES CATTLE CLONING, MOLECULAR HEMOLYSINS MOLECULAR SEQUENCE DATA PEPTIDE FRAGMENTS RECOMBINANT PROTEINS POLYMERASE CHAIN REACTION SPHINGOMYELINS