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Recombinant expression and predicted structure of parborlysin, a cytolytic protein from the Antarctic heteronemertine Parborlasia corrugatus

Author:
Butala, Matej, Šega, Daniel, Tomc, Blaž, Podlesek, Zdravko, Kem, William R., Küpper, Frithjof C., Turk, Tom
Source:
Toxicon 2015 v.108 pp. 32-37
ISSN:
0041-0101
Subject:
Escherichia coli, RNA, cDNA libraries, complementary DNA, cysteine, gene overexpression, genes, head, hemolysis, hydrophobicity, models, plasmids, proteins, toxins, Antarctic region, Antarctica
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.
Agid:
5451299