Recombinant Expression of α‐Bungarotoxin in Pichia pastoris Facilitates Identification of Mutant Toxins Engineered to Recognize Neuronal Nicotinic Acetylcholine Receptors

A snake venom‐derived α‐neurotoxin, α‐bungarotoxin (αBgtx), is the classic competitive antagonist of nicotinic acetylcholine receptors (nAChRs). The very high specificity and essentially irreversible binding of αBgtx to various nAChRs make αBgtx the prime candidate for studying the molecular determinants of specificity for nAChR‐ligand interactions. To facilitate site‐directed mutagenesis of αBgtx for functional analysis, we have developed a recombinant expression system for αBgtx using the methylotropic yeast Pichia pastoris. A synthetic gene coding for αBgtx was subcloned into an expression vector that directs secretion of the recombinant αBgtx (rBgtx) when stably integrated into the yeast genome. Expression of rBgtx was induced by growth of yeast cultures with methanol as the sole carbon source. The activity of the rBgtx in the cell‐free medium was measured by competition with 125I‐Bgtx for binding to Torpedo nAChR‐enriched membranes. The rBgtx, purified to homogeneity by standard HPLC, has the correct predicted amino terminal sequence and molecular mass. Its circular dichroism spectrum is very similar to that of authentic venom‐derived αBgtx, and the biological activity of the rBgtx is identical to that of authentic αBgtx. We have used the Pichia expression system to study a double point mutation of αBgtx, rBgtx‐K38P/L42Q, that has a high affinity for α3β2 neuronal nAChRs. This is the first demonstration of engineering an α‐neurotoxin to recognize non‐α7 neuronal nicotinic receptors.