Recombinant expression of margatoxin and agitoxin-2 in Pichia pastoris: an efficient method for production of KV1.3 channel blockers

Anangi, Raveendra, Koshy, Shyny, Huq, Redwan, Beeton, Christine, Chuang, Woei-Jer and King, Glenn F. (2012) Recombinant expression of margatoxin and agitoxin-2 in Pichia pastoris: an efficient method for production of KV1.3 channel blockers. PLoS One, 7 12: e52965.1-e52965.9. doi:10.1371/journal.pone.0052965


Author Anangi, Raveendra
Koshy, Shyny
Huq, Redwan
Beeton, Christine
Chuang, Woei-Jer
King, Glenn F.
Title Recombinant expression of margatoxin and agitoxin-2 in Pichia pastoris: an efficient method for production of KV1.3 channel blockers
Formatted title
Recombinant expression of margatoxin and agitoxin-2 in Pichia pastoris: an efficient method for production of KV1.3 channel blockers
Journal name PLoS One   Check publisher's open access policy
ISSN 1932-6203
Publication date 2012-12-26
Sub-type Article (original research)
DOI 10.1371/journal.pone.0052965
Open Access Status DOI
Volume 7
Issue 12
Start page e52965.1
End page e52965.9
Total pages 9
Place of publication San Francisco, CA, United States
Publisher Public Library of Science
Collection year 2013
Language eng
Formatted abstract
The Kv1.3 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 Kv1.3 blockers have potential for treatment of autoimmune diseases. Several Kv1.3 channel
blockers have been characterized from scorpion venom, all of which have an a/b 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 Kv1.3 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 Kv1.3 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 KV1.3
(IC50 values of 201639 pM and 9763 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 KV1.3 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 Kv1.3 blockers with therapeutic
potential.
Keyword Agitoxin 2
Disulfide
Hybrid protein
Margatoxin
Q-Index Code C1
Q-Index Status Confirmed Code
Institutional Status UQ

Document type: Journal Article
Sub-type: Article (original research)
Collections: Official 2013 Collection
Institute for Molecular Bioscience - Publications
 
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Created: Mon, 21 Jan 2013, 08:53:41 EST by Susan Allen on behalf of Institute for Molecular Bioscience