title: Recombinant expression of alpha-bungarotoxin in Pichia pastoris facilitates identification of mutant toxins engineered to recognize neuronal nicotinic acetylcholine receptors.
abstract: A snake venom-derived alpha-neurotoxin, alpha-bungarotoxin (alphaBgtx), is the classic competitive antagonist of nicotinic acetylcholine 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 yeast Pichia pastoris. 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 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 1251-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 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-K38P/L42Q, 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.
authors.affiliation: Department of Molecular Pharmacology, Physiology, and Biotechnology, Brown University, Providence, Rhode Island 02912, USA.
authors.forename: M M
authors.initials: MM
authors.lastname: Levandoski
authors.forename: P M
authors.initials: PM
authors.lastname: Caffery
authors.forename: R S
authors.initials: RS
authors.lastname: Rogowski
authors.forename: Y
authors.initials: Y
authors.lastname: Lin
authors.forename: Q L
authors.initials: QL
authors.lastname: Shi
authors.forename: E
authors.initials: E
authors.lastname: Hawrot
mesh_terms: D000818:Animals; D001709:Biotechnology; D002038:Bungarotoxins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009474:Neurons / Q000378:metabolism*; D010843:Pichia / Q000378:metabolism*; D017354:Point Mutation / Q000502:physiology*; D011978:Receptors, Nicotinic / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D014101:Torpedo
chemical_list: D002038:Bungarotoxins; D011978:Receptors, Nicotinic; D011994:Recombinant Proteins