Journal
TOXICON
Volume 108, Issue -, Pages 32-37Publisher
PERGAMON-ELSEVIER SCIENCE LTD
DOI: 10.1016/j.toxicon.2015.09.044
Keywords
Cytolysin; Nemertean; Nemertine; Nucleotide sequence; Parborlasia corrugatus; Parborlysin; Toxin
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Funding
- Collaborative Gearing Scheme of the UK Natural Environment Research Council/British Antarctic Survey [CGS-70]
- Slovenian Research Agency [P1-0207]
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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 similar to 10 kDa basic (pI similar to 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. (C) 2015 Elsevier Ltd. All rights reserved.
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