Production Of EGFP Tagged Vm24 Peptide Toxin: A Fluorescent Probe Of Voltage-Gated Potassuim Channel Kv1.3

Abstract

Venomous peptides identified in various animal species are being directed toward the treatment of T-cell mediated autoimmune disorders such as rheumatoid arthritis, systemic lupus erythematosus, and multiple sclerosis. The selectivity of the venomous peptides can be harnessed in the form of a molecular tool for various research applications by conjugating the peptide to trackable fluorescent proteins. A 36-residues long peptide toxin, Vm24, was purified from the venom of the scorpion Vaejovis mexicanus smithi which has a high affinity for Kv1.3 channels in human lymphocytes (Kd= 2.9 pM) and increased selectivity (>1500-fold) over other ion channels tested. In our approach, an N-terminal His6 tagged EGFP fluorescent protein is conjugated to Vm24 toxin and is expressed in Pichia pastoris. The Pichia clones were screened against a progressively increasing Zeocin™ concentration in search of hyper-resistant clone against Zeocin. and the Conjugate EGPF-Vm24 protein was purified using Ni+ affinity chromatography then further refined using reverse-phase HPLC. The activity of the conjugate was tested through electrophysiological recordings on activated human peripheral T lymphocytes using the whole-cell patch-clamp technique, the chimera protein retained the blocking activity towards Kv1.3 in picomolar range (Kd = 328 pM).