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 (T-EM). Selective blockers constitute valuable therapeutic leads for the treatment of autoimmune diseases mediated by T-EM 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 N-15-labelled ShK were similar to those reported previously for the unlabelled synthetic peptide, confirming that recombinant ShK was correctly folded. Recombinant ShK blocked Kv13 channels with a K-d of 25 pM and inhibited the proliferation of human and rat T lymphocytes with a preference for T-EM cells, with similar potency to synthetic ShK in all assays. This expression system also enables the efficient production of N-15-labelled ShK for NMR studies of peptide dynamics and of the interaction of ShK with Kv1.3 channels. (C) 2012 Elsevier Ltd. All rights reserved.
|Pages (from-to)||840 - 850|
|Number of pages||11|
|Journal||Toxicon : official journal of the International Society on Toxinology|
|Publication status||Published - 2012|