Straightforward approach to produce recombinant scorpion toxins—Pore blockers of potassium channels

Fluorescent Ligands on the Basis of Hongotoxin 1: eGFP-Hongotoxin 1

“…We carried out detailed studies to characterize ability of different Kv1-channel blockers belonging to α-KTx family of scorpion venom -derived peptides to displace eGFP-HgTx1 from the complexes with KcsA-Kv1.3 in a competitive binding assay (Fig.1 C). For this, peptide toxins HgTx1, agitoxin 2 (AgTx2), kaliotoxin (KTX), and charybdotoxin (ChTx), which were obtained in a recombinant form according to the previously developed technique [5], were used. All these peptides exhibited high affinity to the receptor KcsA-Kv1.3 protein, and apparent dissociation constants (Ki) were determined to be: 200 pM for HgTx1, 245 pM for AgTx2, 115 pM for ChTx, and 20 pM for KTX.…”

Scorpion toxins interact with nicotinic acetylcholine receptors

“…Recombinant hongotoxin-1 from the bark scorpion Centruroides limbatus (Hgtx; Kalium name a-KTx 2.5; UniProt ID P59847), kaliotoxin-1 from the fat-tailed scorpion Androctonus mauritanicus (Ktx; Kalium name a-KTx 3.1; UniProt ID P24662), and agitoxin-2 from L. quinquestriatus (AgTx-2; Kalium name a-KTx 3.2; UniProt ID P46111) were produced in E. coli Rosetta-gami(DE3)pLysS (Novagen, Merck KGaA, Darmstadt, Germany) according to [22]. Briefly, in this case the peptide-encoding sequences were cloned at Kpn1 and HindIII restriction sites into the vector pET-23d (Novagen) also containing the MalE gene and a linker sequence from pET-32b as described [22]. Expression was induced with IPTG, the peptides were produced as fusion proteins with maltose-binding protein (MBP), and in this case a TEV protease cleavage site immediately preceded the target sequences.…”

Recombinant scorpion toxins: Focus on four-disulfide peptide blockers of Kv1-channels

“…Recently we have developed an effective and robust bioengineering approach to high-level production (the yields of 12–22 mg per liter of E.coli culture) of fully folded and functionally active recombinant peptides with three disulfide bonds from α-KTx family of scorpion toxins. 2 The approach is based on bacterial expression of the target peptide fused to maltose binding protein (MBP) and subsequent hydrolysis of the fusion protein with tobacco etch virus (TEV) protease. Under optimized expression conditions MBP fusion proteins were over-expressed in the soluble form (>90%) in E.coli cytoplasm with yields about 180–350 mg/l of culture.…”

“…Aiming to extend the developed approach 2 to production of recombinant peptides with 4 disulfide bonds such as hetlaxin (HTX) 3 and maurotoxin (MTX) 4 (both from α-KTx 6 subfamily), we constructed plasmids pET-23d-MalE-HTX and pET-23d-MalE-MTX for expression of the target peptides. It was done using the expression vector pET-23d-MalE, which was described previously.…”

“…It was done using the expression vector pET-23d-MalE, which was described previously. 2 DNA fragments encoding HTX or MTX flanked by N-terminal TEV cleavage site (CS TEV ) were obtained by a polymerase chain reaction using synthetic oligonucleotides, and then each fragment was cloned into the expression vector as described earlier. 2 …”