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Recombinant Expression of Margatoxin and Agitoxin-2 in Pichia pastoris: An Efficient Method for Production of K(V)1.3 Channel Blockers
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...
Autores principales: | , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2012
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3530466/ https://www.ncbi.nlm.nih.gov/pubmed/23300835 http://dx.doi.org/10.1371/journal.pone.0052965 |
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author | Anangi, Raveendra Koshy, Shyny Huq, Redwan Beeton, Christine Chuang, Woei-Jer King, Glenn F. |
author_facet | Anangi, Raveendra Koshy, Shyny Huq, Redwan Beeton, Christine Chuang, Woei-Jer King, Glenn F. |
author_sort | Anangi, Raveendra |
collection | PubMed |
description | 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. |
format | Online Article Text |
id | pubmed-3530466 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2012 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-35304662013-01-08 Recombinant Expression of Margatoxin and Agitoxin-2 in Pichia pastoris: An Efficient Method for Production of K(V)1.3 Channel Blockers Anangi, Raveendra Koshy, Shyny Huq, Redwan Beeton, Christine Chuang, Woei-Jer King, Glenn F. PLoS One Research Article 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. Public Library of Science 2012-12-26 /pmc/articles/PMC3530466/ /pubmed/23300835 http://dx.doi.org/10.1371/journal.pone.0052965 Text en © 2012 Anangi et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited. |
spellingShingle | Research Article Anangi, Raveendra Koshy, Shyny Huq, Redwan Beeton, Christine Chuang, Woei-Jer King, Glenn F. Recombinant Expression of Margatoxin and Agitoxin-2 in Pichia pastoris: An Efficient Method for Production of K(V)1.3 Channel Blockers |
title | Recombinant Expression of Margatoxin and Agitoxin-2 in Pichia pastoris: An Efficient Method for Production of K(V)1.3 Channel Blockers |
title_full | Recombinant Expression of Margatoxin and Agitoxin-2 in Pichia pastoris: An Efficient Method for Production of K(V)1.3 Channel Blockers |
title_fullStr | Recombinant Expression of Margatoxin and Agitoxin-2 in Pichia pastoris: An Efficient Method for Production of K(V)1.3 Channel Blockers |
title_full_unstemmed | Recombinant Expression of Margatoxin and Agitoxin-2 in Pichia pastoris: An Efficient Method for Production of K(V)1.3 Channel Blockers |
title_short | Recombinant Expression of Margatoxin and Agitoxin-2 in Pichia pastoris: An Efficient Method for Production of K(V)1.3 Channel Blockers |
title_sort | recombinant expression of margatoxin and agitoxin-2 in pichia pastoris: an efficient method for production of k(v)1.3 channel blockers |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3530466/ https://www.ncbi.nlm.nih.gov/pubmed/23300835 http://dx.doi.org/10.1371/journal.pone.0052965 |
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