Expression of a recombinant Phoneutria toxin active in calcium channels.
PnTx3-4 is a toxin isolated from the venom of the spider Phoneutria nigriventer that blocks N-, P/Q-, and R-type voltage-gated calcium channels and has great potential for clinical applications. In this report we used the SUMO system to express large amounts of recombinant PnTx3-4 peptide, which was found in both soluble and insoluble fractions of bacterial extracts. We purified the recombinant toxin from both fractions and showed that the recombinant peptide showed biological activity similar to the native PnTx3-4. In silico analysis of the primary sequence of PnTx3-4 indicated that the peptide conforms to all the criteria of a knottin scaffold. Additionally, circular dichroism spectrum analysis of the recombinant PnTx3-4 predicted that the toxin structure is composed of approximately 53% turns/unordered, 31% α-helix and 16% β-strand, which is consistent with predicted model of the PnTx3-4 knottin scaffold available at the knottin database (http://knottin.cbs.cnrs.fr). These studies provide the basis for future large scale production and structure-function investigation of PnTx3-4.
Molecular Brain Research Group, Robarts Research Institute, Department of Anatomy & Cell Biology, Schulich School of Medicine & Dentistry, University of Western Ontario, Canada.
I A
IA
Souza
E A
EA
Cino
W Y
WY
Choy
M N
MN
Cordeiro
M
M
Richardson
C
C
Chavez-Olortegui
M V
MV
Gomez
M A M
MA
Prado
V F
VF
Prado
D000595:Amino Acid Sequence; D000704:Analysis of Variance; D000818:Animals; D015220:Calcium Channels / Q000378:metabolism*; D002942:Circular Dichroism; D008969:Molecular Sequence Data; D009479:Neuropeptides / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D009841:Oligonucleotides / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D017510:Protein Folding; D011994:Recombinant Proteins / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D013111:Spider Venoms / Q000378:metabolism*; D013574:Synaptosomes / Q000378:metabolism
D015220:Calcium Channels; D009479:Neuropeptides; D009841:Oligonucleotides; D011994:Recombinant Proteins; D013111:Spider Venoms; C098736:Tx3 neurotoxin