title: Recombinant expression of margatoxin and agitoxin-2 in Pichia pastoris: an efficient method for production of KV1.3 channel blockers.
abstract: The K(v)1.3 voltage-gated potassium channel regulates membrane potential and calcium signaling in human effector memory T cells that are key mediators of autoimmune diseases such as multiple sclerosis, type 1 diabetes, and rheumatoid arthritis. Thus, subtype-specific K(v)1.3 blockers have potential for treatment of autoimmune diseases. Several K(v)1.3 channel blockers have been characterized from scorpion venom, all of which have an α/β scaffold stabilized by 3-4 intramolecular disulfide bridges. Chemical synthesis is commonly used for producing these disulfide-rich peptides but this approach is time consuming and not cost effective for production of mutants, fusion proteins, fluorescently tagged toxins, or isotopically labelled peptides for NMR studies. Recombinant production of K(v)1.3 blockers in the cytoplasm of E. coli generally necessitates oxidative refolding of the peptides in order to form their native disulfide architecture. An alternative approach that avoids the need for refolding is expression of peptides in the periplasm of E. coli but this often produces low yields. Thus, we developed an efficient Pichia pastoris expression system for production of K(v)1.3 blockers using margatoxin (MgTx) and agitoxin-2 (AgTx2) as prototypic examples. The Pichia system enabled these toxins to be obtained in high yield (12-18 mg/L). NMR experiments revealed that the recombinant toxins adopt their native fold without the need for refolding, and electrophysiological recordings demonstrated that they are almost equipotent with the native toxins in blocking K(V)1.3 (IC(50) values of 201±39 pM and 97 ± 3 pM for recombinant AgTx2 and MgTx, respectively). Furthermore, both recombinant toxins inhibited T-lymphocyte proliferation. A MgTx mutant in which the key pharmacophore residue K28 was mutated to alanine was ineffective at blocking K(V)1.3 and it failed to inhibit T-lymphocyte proliferation. Thus, the approach described here provides an efficient method of producing toxin mutants with a view to engineering K(v)1.3 blockers with therapeutic potential.
authors.affiliation: Institute for Molecular Bioscience, The University of Queensland, St Lucia, Queensland, Australia. r.anangi@imb.uq.edu.au
authors.forename: Raveendra
authors.initials: R
authors.lastname: Anangi
authors.forename: Shyny
authors.initials: S
authors.lastname: Koshy
authors.forename: Redwan
authors.initials: R
authors.lastname: Huq
authors.forename: Christine
authors.initials: C
authors.lastname: Beeton
authors.forename: Woei-Jer
authors.initials: WJ
authors.lastname: Chuang
authors.forename: Glenn F
authors.initials: GF
authors.lastname: King
mesh_terms: D000818:Animals; D002460:Cell Line; D049109:Cell Proliferation; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D051379:Mice; D010843:Pichia / Q000378:metabolism*; D026902:Potassium Channel Blockers / Q000138:chemical synthesis*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000138:chemical synthesis* / Q000378:metabolism; D012604:Scorpion Venoms / Q000138:chemical synthesis* / Q000378:metabolism; D014118:Toxins, Biological / Q000138:chemical synthesis* / Q000378:metabolism
chemical_list: D026902:Potassium Channel Blockers; D011994:Recombinant Proteins; D012604:Scorpion Venoms; D014118:Toxins, Biological; C087603:agitoxin 2; C077910:margatoxin