title: Recombinant expression and in vitro characterisation of active Huwentoxin-IV.
abstract: Huwentoxin-IV (HwTx-IV) is a 35-residue neurotoxin peptide with potential application as a novel analgesic. It is a member of the inhibitory cystine knot (ICK) peptide family, characterised by a compact globular structure maintained by three intramolecular disulfide bonds. Here we describe a novel strategy for producing non-tagged, fully folded ICK-toxin in a bacterial system. HwTx-IV was expressed as a cleavable fusion to small ubiquitin-related modifier (SUMO) in the cytoplasm of the SHuffle T7 Express lysY Escherichia coli strain, which allows cytosolic disulfide bond formation. Purification by IMAC with selective elution of monomeric SUMO fusion followed by proteolytic cleavage and polishing chromatographic steps yielded pure homogeneous toxin. Recombinant HwTx-IV is produced with a C-terminal acid, whereas the native peptide is C-terminally amidated. HwTx-IV(acid) inhibited Nav1.7 in a dose dependent manner (IC50 = 463-727 nM). In comparison to HwTx-IV(amide) (IC50 = 11 ± 3 nM), the carboxylate was ~50 fold less potent on Nav1.7, which highlights the impact of the C-terminus. As the amide bond of an additional amino acid may mimic the carboxamide, we expressed the glycine-extended analogue HwTx-IV(G36)(acid) in the SUMO/SHuffle system. The peptide was approximately three fold more potent on Nav1.7 in comparison to HwTx-IV(acid) (IC50 = 190 nM). In conclusion, we have established a novel system for expression and purification of fully folded and active HwTx-IV(acid) in bacteria, which could be applicable to other structurally complex and cysteine rich peptides. Furthermore, we discovered that glycine extension of HwTx-IV(acid) restores some of the potency of the native carboxamide. This finding may also apply to other C-terminally amidated peptides produced recombinantly.
authors: Antibody Discovery and Protein Engineering, Research, MedImmune, Cambridge, United Kingdom.
authors: Isabelle
authors: I
authors: Sermadiras
authors: Jefferson
authors: J
authors: Revell
authors: John E
authors: JE
authors: Linley
authors: Alan
authors: A
authors: Sandercock
authors: Peter
authors: P
authors: Ravn
mesh_terms: D000595:Amino Acid Sequence; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D015870:Gene Expression; D005998:Glycine / Q000737:chemistry; D006801:Humans; D008958:Models, Molecular; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013111:Spider Venoms / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D011993:Recombinant Fusion Proteins; D013111:Spider Venoms; C470632:huwentoxin IV, Selenocosmia huwena; D005998:Glycine