Recombinant expression of alpha-bungarotoxin in XXXX facilitates identification of mutant toxins engineered to recognize neuronal nicotinic @entity934 receptors.

@entity72752 @entity1472 @entity10056 @entity1042 @entity72753 @entity505 @entity375 @entity934
A snake venom-derived alpha-neurotoxin, alpha-bungarotoxin (alphaBgtx), is the classic competitive antagonist of nicotinic @entity934 receptors (nAChRs). The very high specificity and essentially irreversible binding of alphaBgtx to various nAChRs make alphaBgtx the prime candidate for studying the molecular determinants of specificity for nAChR-ligand interactions. To facilitate site-directed mutagenesis of alphaBgtx for functional analysis, we have developed a recombinant expression system for alphaBgtx using the methylotropic @entity375 @entity10056 . A synthetic gene coding for alphaBgtx was subcloned into an expression vector that directs secretion of the recombinant alphaBgtx (rBgtx) when stably integrated into the @entity375 genome. Expression of rBgtx was induced by growth of @entity375 cultures with @entity1042 as the sole @entity505 source. The activity of the rBgtx in the cell-free medium was measured by competition with 1251-Bgtx for binding to Torpedo nAChR-enriched membranes. The rBgtx, purified to homogeneity by standard HPLC, has the correct predicted @entity1472 terminal sequence and molecular mass. Its circular dichroism spectrum is very similar to that of authentic venom-derived alphaBgtx, and the biological activity of the rBgtx is identical to that of authentic alphaBgtx. We have used the Pichia expression system to study a double point mutation of alphaBgtx, rBgtx- @entity72752 / @entity72753 , that has a high affinity for alpha3beta2 neuronal nAChRs. This is the first demonstration of engineering an alpha-neurotoxin to recognize non-alpha7 neuronal nicotinic receptors.
