title: Recombinant expression and predicted structure of parborlysin, a cytolytic protein from the Antarctic heteronemertine Parborlasia corrugatus.
abstract: The heteronemertine Parborlasia corrugatus contains a cytolytic protein, parborlysin, which after extensive purification was found by Edman sequencing to be a mixture of several homologues. To investigate this microheterogeneity and enable the analysis of single toxins, we have obtained seven parborlysin isoform genes from P. corrugatus collected in Antarctica. Total RNA was isolated from the homogenized head region and parborlysin genes were identified from a cDNA library using degenerate primers. The translated sequences reveal that the isoforms are ∼ 10 kDa basic (pI ∼ 10) proteins of which all but one harbour six cysteine residues. We generated a model of the three dimensional structure of parborlysins, which suggests that they are composed of five alpha-helical segments that include large, exposed hydrophobic surfaces. Finally, we constructed plasmids and inserted them into Escherichia coli to obtain overexpressed amino- or carboxy-terminal polyhistidine-tagged parborlysin isoforms fused to the third domain of the E. coli periplasmic-protein TolA to facilitate toxin isolation. One of the isoforms adversely affected growth in the E. coli expressing it. Although we succeeded in isolating one of the recombinant parborlysin constructs, it lacked haemolytic activity.
authors.affiliation: Department of Biology, Biotechnical Faculty, University of Ljubljana, Ljubljana, Slovenia.
authors.forename: Matej
authors.initials: M
authors.lastname: Butala
authors.affiliation: Department of Biology, Biotechnical Faculty, University of Ljubljana, Ljubljana, Slovenia.
authors.forename: Daniel
authors.initials: D
authors.lastname: Šega
authors.affiliation: Department of Biology, Biotechnical Faculty, University of Ljubljana, Ljubljana, Slovenia.
authors.forename: Blaž
authors.initials: B
authors.lastname: Tomc
authors.affiliation: Department of Biology, Biotechnical Faculty, University of Ljubljana, Ljubljana, Slovenia.
authors.forename: Zdravko
authors.initials: Z
authors.lastname: Podlesek
authors.affiliation: Department of Pharmacology and Therapeutics, College of Medicine, University of Florida, Gainesville, FL, 32610, USA.
authors.forename: William R
authors.initials: WR
authors.lastname: Kem
authors.affiliation: Oceanlab, University of Aberdeen, Main Street, Newburgh, AB41 6AA, Scotland, UK.
authors.forename: Frithjof C
authors.initials: FC
authors.lastname: Küpper
authors.affiliation: Department of Biology, Biotechnical Faculty, University of Ljubljana, Ljubljana, Slovenia. Electronic address: tom.turk@bf.uni-lj.si.
authors.forename: Tom
authors.initials: T
authors.lastname: Turk
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D007448:Invertebrates / Q000235:genetics* / Q000378:metabolism; D008387:Marine Toxins / Q000737:chemistry* / Q000235:genetics / Q000633:toxicity; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism; D016415:Sequence Alignment; D020539:Sequence Analysis, Protein
chemical_list: D008387:Marine Toxins; D011994:Recombinant Proteins
keywords: Cytolysin; Nemertean; Nemertine; Nucleotide sequence; Parborlasia corrugatus; Parborlysin; Toxin