Expression and isotopic labelling of the potassium channel blocker ShK toxin as a thioredoxin fusion protein in bacteria.
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 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 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 ¹⁵N-labelled ShK for NMR studies of peptide dynamics and of the interaction of ShK with Kv1.3 channels.
Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria 3052, Australia.
Shih Chieh
SC
Chang
Charles A
CA
Galea
Eleanor W W
EW
Leung
Rajeev B
RB
Tajhya
Christine
C
Beeton
Michael W
MW
Pennington
Raymond S
RS
Norton
D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D003064:Cnidarian Venoms / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli; D007553:Isotope Labeling; D008958:Models, Molecular; D009587:Nitrogen Isotopes / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular; D018408:Patch-Clamp Techniques; D016133:Polymerase Chain Reaction; D026902:Potassium Channel Blockers / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D011487:Protein Conformation; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000378:metabolism* / Q000494:pharmacology; D013601:T-Lymphocytes / Q000187:drug effects; D013879:Thioredoxins / Q000378:metabolism* / Q000494:pharmacology
D003064:Cnidarian Venoms; D017931:DNA Primers; D009587:Nitrogen Isotopes; D026902:Potassium Channel Blockers; D011993:Recombinant Fusion Proteins; C095391:ShK neurotoxin; D013879:Thioredoxins