title: Gene construction, expression and functional testing of an inotropic peptide from the venom of the black scorpion Hottentotta judaicus.
abstract: 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.
authors.affiliation: Institut für Pharmakologie und Toxikologie, Universitätsklinikum Münster, Domagkstr. 12, D-48149 Münster, Germany.
authors.forename: M A
authors.initials: MA
authors.lastname: Tekook
authors.forename: L
authors.initials: L
authors.lastname: Fabritz
authors.forename: P
authors.initials: P
authors.lastname: Kirchhof
authors.forename: S
authors.initials: S
authors.lastname: König
authors.forename: F U
authors.initials: FU
authors.lastname: Müller
authors.forename: W
authors.initials: W
authors.lastname: Schmitz
authors.forename: T
authors.initials: T
authors.lastname: Tal
authors.forename: E
authors.initials: E
authors.lastname: Zlotkin
authors.forename: U
authors.initials: U
authors.lastname: Kirchhefer
mesh_terms: D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004247:DNA; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D013058:Mass Spectrometry; D016296:Mutagenesis; D010455:Peptides / Q000235:genetics* / Q000633:toxicity*; D016133:Polymerase Chain Reaction; D012604:Scorpion Venoms / Q000737:chemistry*; D012605:Scorpions; D012689:Sequence Homology, Nucleic Acid
chemical_list: D017931:DNA Primers; D010455:Peptides; D012604:Scorpion Venoms; D004247:DNA