Cloning and expression of mamba toxins

Abstract

Mamba venoms contain pharmacologically active proteins that interfere with neuromuscular transmission by binding to and altering the normal functioning of neuronal proteins involved, directly or indirectly, with regulating nerve transmission. Of the mamba toxins studied to date, many act on voltage-sensitive K + channels, nicotinic or muscarinic acetylcholine receptors, or acetylcholinesterase. In an attempt to clone, characterize, and express the genes encoding these toxins, as well as other genes specifying activities not completely elucidated as yet, a cDNA library was constructed from mRNA isolated from the glands of the black mamba. Clones from the library harboring sequences encoding 14 different mamba toxins were isolated and characterized by nucleotide sequence analysis. Genes coding for three proteins, dendrotoxins (DTX) K, I, and E, were expressed as maltose-binding (MBP) fusion proteins in the periplasmic space of Escherichia coli. The DTX K-MBP fusion protein was affinity purified, cleaved from its chaperon, and the recombinant DTX K purified from MBP. Recombinant DTX K was shown to be identical to native DTX K in its N-terminal sequence, Chromatographic behavior, convulsion-inducing activity, and binding to voltage-activated K + channels in bovine synaptic membranes. Computer modeling was employed to create three-dimensional structures of DTX K and DTX I from the X-ray crystal structure of α-DTX utilizing both structural and sequence homologies. Comparisons were made between the three toxins, providing a framework for site-directed mutagenesis.