Expression and isotopic labelling of the potassium channel blocker ShK toxin as a thioredoxin fusion protein in bacteria

Abstract

The polypeptide toxin ShK is a potent blocker of Kv1.3 potassium channels, which play a crucial role in the activation of human effector memory T-cells (TEM). Selective blockers constitute valuable therapeutic leads for the treatment of autoimmune diseases mediated by TEM cells, such as multiple sclerosis, rheumatoid arthritis, and type-1 diabetes. We have established a recombinant peptide expression system in order to generate isotopically-labelled ShK and various ShK analogues for in-depth biophysical and pharmacological studies. ShK was expressed as a thioredoxin fusion protein in Escherichia coli BL21 (DE3) cells and purified initially by Ni2+ iminodiacetic acid affinity chromatography. The fusion protein was cleaved with enterokinase and purified to homogeneity by reverse-phase HPLC. NMR spectra of 15N-labelled ShK were similar to those reported previously for the unlabelled synthetic peptide, confirming that recombinant ShK was correctly folded. Recombinant ShK blocked Kv1.3 channels with a Kd of 25 pM and inhibited the proliferation of human and rat T lymphocytes with a preference for TEM cells, with similar potency to synthetic ShK in all assays. This expression system also enables the efficient production of 15N-labelled ShK for NMR studies of peptide dynamics and of the interaction of ShK with Kv1.3 channels.

Prolonged immunomodulation in inflammatory arthritis using the selective Kv1.3 channel blocker HsTX1[R14A] and its PEGylated analog

“…For example, fusing OsK1, OdK2, or ShK with Fc fragments of immunoglobulins, with PEG moieties, or with albumin reduced their affinity for Kv1.3 channels to varying degrees [40; 63]. A thioredoxin fusion of ShK or a biotin-tagged ShK bound to streptavidin had no effect on Kv1.3 currents at 100 nM, whereas ShK itself blocks the channel with low picomolar affinity [44; 64]. …”

Conformational Flexibility in the Binding Surface of the Potassium Channel Blocker ShK

“…In particular, the behavior of small polypeptides such as ShK is best described as a combination of several stable conformations in equilibrium, and understanding the dynamics of inter-conversions within this conformational space is a prerequisite for a full picture of their binding capabilities. [21] The availability of recombinantly labeled ShK [12] allows its motions to be mapped using NMR spectroscopy by measuring relaxation rates of 15 N nuclei along the toxin backbone.…”

“…[12] Lyophilized 15 N- or 13 C, 15 N-labeled ShK was dissolved in 10 mM phosphate buffer (adjusted to pH 5.4 or 7.0) containing 10 mM NaCl and 7 % 2 H 2 O. Typical ShK concentrations were 0.3-0.9 mM.…”

“…Recently, an efficient system for expressing correctly folded and biologically active ShK in isotopically-labeled form has been developed. [12] Here we employ 15 N-spin relaxation measurements to characterize structural dynamics of the ShK backbone. These data show that ShK behaves as a close-to-spherical polypeptide with a global tumbling time of 2.6 ns at 293 K, and exhibits backbone rigidity on the ps-ns timescale for residues 3-35.…”

CHAPTER 10. Case Study 2: Transforming a Toxin into a Therapeutic: the Sea Anemone Potassium Channel Blocker ShK Toxin for Treatment of Autoimmune Diseases

“…18,37 ShK-186 inhibited production of the cytokines IL-2 and IFNg by CCR7 -T EM lymphocytes more efficiently than in the case of CCR7 + naïve/T CM lymphocytes. 18 It was 17,18,24,[27][28][29][30]33,37,39,41 signicantly less effective in inhibiting production of IL-4 and TNFa. ShK-186 persistently inhibited the proliferation of CCR7 -T EM lymphocytes (IC 50 100 pM) whereas CCR7 + naïve/T CM lymphocytes were 10-fold less sensitive to the blocker at rst, and became completely resistant to ShK-186 aer their upregulation of the KCa3.1 channel.…”