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

• Published in: Toxicon (Oxford) 2012-12, Vol.60 (8), p.1415-1427
• Main Authors: Tekook, M.A., Fabritz, L., Kirchhof, P., König, S., Müller, F.U., Schmitz, W., Tal, T., Zlotkin, E., Kirchhefer, U.
• Format: Article
• Language: English
• Subjects: additives; agarose; amino acids; Animal poisons toxicology. Antivenoms; Animals; Base Sequence; bioactive properties; Biological and medical sciences; Blotting, Western; Cardiomyocyte contraction; cardiomyocytes; Chromatography, Affinity; Chromatography, High Pressure Liquid; Cloning, Molecular; disulfide bonds; DNA; DNA Primers; Electrophoresis, Polyacrylamide Gel; Escherichia coli; Escherichia coli - genetics; genes; glutathione; glycerol; high performance liquid chromatography; HPLC; insect larvae; In vitro refolding; Mass Spectrometry; Medical sciences; mice; Mutagenesis; nucleotide sequences; paralysis; Peptides - genetics; Peptides - toxicity; Polymerase Chain Reaction; polypeptides; Positive inotropy; Prokaryotic expression; Scorpion Venoms - chemistry; Scorpiones; Scorpions; Sequence Homology, Nucleic Acid; sodium channels; Toxicology; toxins; venoms; βNaScTx
• ISSN: 0041-0101; 1879-3150
• DOI: 10.1016/j.toxicon.2012.10.008

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 (C18) 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. ► Bj-IP, a new anti-insect depressant βNaScTx, was isolated from Hottentotta judaicus. ► The synthesis of this toxin gene was achieved by splicing by overlap extension PCR. ► Bj-IP was expressed in Escherichia coli and purified by NiNTA agarose and HPLC. ► Mass spectrometry confirmed the theoretical mass of the recombinant toxin (6608 Da). ► After refolding, Bj-IP increased myocyte contraction and accelerat