Molecular cloning and functional expression of hemolysin from the sea anemone Actineria villosa.
The full-length cDNA that encodes the hemolytic toxin Avt-I, with 226 amino acids, from the venomous sea anemone Actineria villosa has been cloned using the oligo-capping method. The cDNA contains 681bp open reading frame and its predicted amino acid sequences revealed that Avt-I was basic polypeptides without cysteine residues and Arg-Gly-Asp (RGD) motif sequence. The mature Avt-I has a predicted molecular weight of 19.6 kDa and its theoretical isoelectric point is 9.3. The Avt-I revealed 99, 61, 57, and 57% amino acid similarity with hemolytic toxins Pstx20, EqtII, StII, and HmT from Phyllodiscus semoni, Actinia Equina, Stichodactyla helianthus, and Heteractis magnifica, respectively. The characteristic amphiphilic alpha-helix structure was found at the N-terminal region of the mature Avt-I. Recombinant Avt-I (rAvt-I) was expressed in Escherichia coli BL21 (DE3) strain as a biologically active form and purified rAvt-I caused 50% hemolytic activity against 1% sheep erythrocytes at a concentration of 6.3 ng/ml (0.32 nM). M9Y medium led to more than 2-fold increase in rAvt-I yield than cultivation in Luria-Bertani medium.
Uechi
Gen-ichiro
G
Division of Tropical Parasitology, Unit of Social and Environmental Medicine, School of Medicine, University of the Ryukyus, Nishihara, Okinawa 903-0215, Japan. k018761@eve.u-ryukyu.ac.jp
Toma
Hiromu
H
Arakawa
Takeshi
T
Sato
Yoshiya
Y
D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D018076:DNA, Complementary / Q000235:genetics; D004912:Erythrocytes / Q000187:drug effects; D004926:Escherichia coli / Q000235:genetics; D006460:Hemolysin Proteins / Q000737:chemistry / Q000235:genetics* / Q000494:pharmacology; D007526:Isoelectric Point; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000235:genetics; D012615:Sea Anemones / Q000737:chemistry*; D017385:Sequence Homology; D012756:Sheep
D018076:DNA, Complementary; D006460:Hemolysin Proteins; D011994:Recombinant Proteins