Recombinant Expression of Margatoxin and Agitoxin-2 in Pichia pastoris: An Efficient Method for Production of KV1.3 Channel Blockers

Raveendra Anangi, Shyny Koshy, Redwan Huq, Christine Beeton, Woei Jer Chuang, Glenn F. King

Research output: Contribution to journalArticle

9 Citations (Scopus)

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 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 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 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 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.

Original languageEnglish
Article numbere52965
JournalPloS one
Volume7
Issue number12
DOIs
Publication statusPublished - 2012 Dec 29

Fingerprint

Pichia pastoris
Pichia
T-cells
toxins
Disulfides
Peptides
T-Lymphocytes
sulfides
peptides
Escherichia coli
Autoimmune Diseases
Kv1.3 Potassium Channel
T-lymphocytes
autoimmune diseases
Nuclear magnetic resonance
lymphocyte proliferation
Scorpion Venoms
Voltage-Gated Potassium Channels
Periplasm
mutants

All Science Journal Classification (ASJC) codes

  • Biochemistry, Genetics and Molecular Biology(all)
  • Agricultural and Biological Sciences(all)
  • General

Cite this

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 KV1.3 Channel Blockers. In: PloS one. 2012 ; Vol. 7, No. 12.
@article{2c3cb4c029e949d492e06610b5db6480,
title = "Recombinant Expression of Margatoxin and Agitoxin-2 in Pichia pastoris: An Efficient Method for Production of KV1.3 Channel Blockers",
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 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 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 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 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.",
author = "Raveendra Anangi and Shyny Koshy and Redwan Huq and Christine Beeton and Chuang, {Woei Jer} and King, {Glenn F.}",
year = "2012",
month = "12",
day = "29",
doi = "10.1371/journal.pone.0052965",
language = "English",
volume = "7",
journal = "PLoS One",
issn = "1932-6203",
publisher = "Public Library of Science",
number = "12",

}

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; King, Glenn F.

In: PloS one, Vol. 7, No. 12, e52965, 29.12.2012.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Recombinant Expression of Margatoxin and Agitoxin-2 in Pichia pastoris

T2 - An Efficient Method for Production of KV1.3 Channel Blockers

AU - Anangi, Raveendra

AU - Koshy, Shyny

AU - Huq, Redwan

AU - Beeton, Christine

AU - Chuang, Woei Jer

AU - King, Glenn F.

PY - 2012/12/29

Y1 - 2012/12/29

N2 - 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 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 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 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 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.

AB - 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 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 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 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 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.

UR - http://www.scopus.com/inward/record.url?scp=84871548782&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84871548782&partnerID=8YFLogxK

U2 - 10.1371/journal.pone.0052965

DO - 10.1371/journal.pone.0052965

M3 - Article

C2 - 23300835

AN - SCOPUS:84871548782

VL - 7

JO - PLoS One

JF - PLoS One

SN - 1932-6203

IS - 12

M1 - e52965

ER -