Facile production of native-like kappa-bungarotoxin in yeast: an enhanced system for the production of a neuronal nicotinic acetylcholine receptor probe.
Research on the mammalian central nervous system had been hindered by the limited number and meager supply of naturally occurring toxins that can be used as pharmacological reagents. The kappa-neurotoxins in particular are not found abundantly in nature and are difficult to obtain and isolate in quantities sufficient for research purposes. Here we report the expression and isolation of relatively large quantities of the kappa-neurotoxin, kappa-bungarotoxin, in an active form using a yeast, Pichia pastoris, expression system. The resultant product of the expression system has a short amino-terminal amino acid extension relative to venom-derived kappa-bungarotoxin, but is equivalent to the native toxin in physical and biological properties, as judged by the CD spectra, the ability to form dimers in solution, and the activity on chick ciliary ganglia. The yeast system produces approximately 0.2 mg from a 2 liter culture and the purification takes approximately 2 days. In contrast, E. coli, the only other available expression system for this toxin, produces one-fifth to one-half as much active material from a 5 liter high-density fermentation and the resulting protein takes over a week to purify. No high mol. wt disulfide-bonded aggregates were found in the yeast expression system product, indicating that the product is that of a biologically assisted folding process. This has significant implications not only for the efficient production of native toxin but also for the production of mutant proteins to study the structure-function relationship in these proteins.
Fiordalisi
J J
JJ
Department of Molecular Biology and Pharmacology, Washington University School of Medicine, St Louis, MO 63110, USA.
James
P L
PL
Zhang
Y
Y
Grant
G A
GA
D000109:Acetylcholine / Q000494:pharmacology*; D000595:Amino Acid Sequence; D001483:Base Sequence; D002038:Bungarotoxins / Q000096:biosynthesis* / Q000737:chemistry / Q000633:toxicity; D002852:Chromatography, Ion Exchange; D015870:Gene Expression / Q000235:genetics; D008969:Molecular Sequence Data; D009474:Neurons / Q000378:metabolism*; D016133:Polymerase Chain Reaction / Q000379:methods; D011978:Receptors, Nicotinic / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry; D015003:Yeasts / Q000378:metabolism*
D002038:Bungarotoxins; D011978:Receptors, Nicotinic; D011993:Recombinant Fusion Proteins; D000109:Acetylcholine