Gene construction, expression and functional testing of an inotropic peptide from the venom of the black scorpion Hottentotta judaicus

M A Tekook, L Fabritz, P Kirchhof, S König, F U Müller, W Schmitz, T Tal, E Zlotkin, U Kirchhefer

Research output: Contribution to journalArticlepeer-review

3 Citations (Scopus)


Anti-insect depressant toxins represent a subfamily of scorpion venom-derived β-toxins that are polypeptides composed of 61-65 amino acid residues stabilized by four disulfide bridges. These toxins affect the activation of voltage-sensitive sodium channels (NaScTx) and exhibit the preferential ability to induce flaccid paralysis in insect larvae. Here we demonstrate the recombinant expression of the novel cardiac inotropic peptide (Bj-IP) that was classified as an anti-insect depressant βNaScTx isolated from the venom of Hottentotta judaicus. By using "splicing by overlap extension" (SOE)-PCR, allowing for the first time one step de novo synthesis of long-chain scorpion toxin genes, we generated a codon-optimized DNA fragment of Bj-IP for cloning into the Escherichia coli vector pQE30. Moreover, the gene of interest was fused to a 6xHis coding DNA sequence. Subsequent recombinant expression was performed in E. coli KRX. The purification of the polypeptide was achieved by a combination of NiNTA agarose columns and RP (C(18)) high-performance liquid chromatography. The purified fusion protein was digested with factor Xa resulting in the elution of Bj-IP. The yield of recombinant Bj-IP expression was approximately 4.5 mg per liter of culture. Mass spectrometry confirmed the theoretical total mass of Bj-IP (6608 Da). Tag-free Bj-IP was refolded in guanidine chloride buffer with a glutathione redox system which was supplemented with different additives at 16 °C. Supplementation with 10% glycerol produced Bj-IP folding forms that exhibited reproducible biological activity in mouse cardiomyocytes. Cell contractility was increased by almost 3-fold and decay kinetics were hasten by 47% after administration of Bj-IP. Taken together, here we show the recombinant expression of the functionally active cardiac inotropic peptide Bj-IP, a new βNaScTx from H. judaicus, for promising pharmacological applications. Furthermore, our data suggest that the use of SOE-PCR may help to facilitate in future the high throughput of cloning and/or modification of scorpion toxin genes.

Original languageEnglish
Pages (from-to)1415-27
Number of pages13
Issue number8
Publication statusPublished - 15 Dec 2012


  • Animals
  • Base Sequence
  • Blotting, Western
  • Chromatography, Affinity
  • Chromatography, High Pressure Liquid
  • Cloning, Molecular
  • DNA
  • DNA Primers
  • Electrophoresis, Polyacrylamide Gel
  • Escherichia coli
  • Mass Spectrometry
  • Mutagenesis
  • Peptides
  • Polymerase Chain Reaction
  • Scorpion Venoms
  • Scorpions
  • Sequence Homology, Nucleic Acid


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