title: A novel ICK peptide from the Loxosceles intermedia (brown spider) venom gland: cloning, heterologous expression and immunological cross-reactivity approaches.
abstract: The venom of a Loxosceles spider is composed of a complex mixture of biologically active components, consisting predominantly of low molecular mass molecules (3-45 kDa). Transcriptome analysis of the Loxosceles intermedia venom gland revealed ESTs with similarity to the previously described LiTx peptides. Sequences similar to the LiTx3 isoform were the most abundant, representing approximately 13.9% of all ESTs and 32% of the toxin-encoding messengers. These peptides are grouped in the ICK (Inhibitor Cystine Knot) family, which contains single chain molecules with low molecular mass (3-10 kDa). Due to their high number of cysteine residues, ICK peptides form intramolecular disulfide bridges. The aims of this study were to clone and express a novel ICK peptide isoform, as well as produce specific hyperimmune serum for immunoassays. The corresponding cDNA was amplified by PCR using specific primers containing restriction sites for the XhoI and BamHI enzymes; this PCR product was then ligated in the pET-14b vector and transformed into E. coli AD494 (DE3) cells. The peptide was expressed by IPTG induction for 4 h at 30 °C and purified by affinity chromatography with Ni-NTA resin. Hyperimmune serum to the recombinant peptide was produced in rabbits and was able to specifically recognize both the purified recombinant peptide and the native form present in the venom. Furthermore, the recombinant peptide was recognized by antisera raised against L. intermedia, L. gaucho and L. laeta whole venoms. The recombinant peptide obtained will enable future studies to characterize its biological activity, as well as investigations regarding possible biotechnological applications.
authors.affiliation: Department of Cell Biology, Federal University of Paraná, Jardim das Américas, 81531-990 Curitiba, Paraná, Brazil.
authors.forename: Fernando Hitomi
authors.initials: FH
authors.lastname: Matsubara
authors.forename: Luiza Helena
authors.initials: LH
authors.lastname: Gremski
authors.forename: Gabriel Otto
authors.initials: GO
authors.lastname: Meissner
authors.forename: Eduardo Soares
authors.initials: ES
authors.lastname: Constantino Lopes
authors.forename: Waldemiro
authors.initials: W
authors.lastname: Gremski
authors.forename: Andrea
authors.initials: A
authors.lastname: Senff-Ribeiro
authors.forename: Olga Meiri
authors.initials: OM
authors.lastname: Chaim
authors.forename: Silvio Sanches
authors.initials: SS
authors.lastname: Veiga
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D003429:Cross Reactions; D015180:Electrophoresis, Gel, Two-Dimensional; D004926:Escherichia coli; D020224:Expressed Sequence Tags; D020869:Gene Expression Profiling; D015151:Immunoblotting; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010455:Peptides / Q000737:chemistry* / Q000235:genetics; D010727:Phosphoric Diester Hydrolases / Q000737:chemistry* / Q000235:genetics; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D013111:Spider Venoms / Q000737:chemistry* / Q000235:genetics; D013112:Spiders / Q000737:chemistry*
chemical_list: D010455:Peptides; D020033:Protein Isoforms; D011994:Recombinant Proteins; D013111:Spider Venoms; C055161:loxosceles venom; D010727:Phosphoric Diester Hydrolases
keywords: ICK peptide; Knottin; Loxosceles intermedia; Spider venom