RESUMEN
The expression of kappa-bungarotoxin in Escherichia coli from a synthetic gene results in the production of multiple species of polypeptide. These include not only biologically active kappa-bungarotoxin but also a variety of inactive species, which include inactive monomers as well as disulfide-linked polymeric species. Identification of these species and their separation from the biologically active recombinant toxin is necessary for the use of the toxin in physiological and biochemical studies. This has been accomplished by a combination of ion-exchange and reverse-phase chromatography which results in a homogeneous toxin preparation. The active material produced is sufficient for many types of biological studies and for mutagenesis experiments directed at determining the structure function relationships of toxin interactions with the neuronal nicotinic acetylcholine receptor. In addition, the kappa-bungarotoxin produced in this manner has the distinct advantage over venom-purified kappa-bungarotoxin of not being contaminated with other venom components which could potentially affect experimental observations.
Asunto(s)
Bungarotoxinas/aislamiento & purificación , Secuencia de Aminoácidos , Aminoácidos/análisis , Animales , Bioensayo , Bungarotoxinas/química , Bungarotoxinas/genética , Dicroismo Circular , Escherichia coli/genética , Datos de Secuencia Molecular , Fragmentos de Péptidos/aislamiento & purificación , Conformación Proteica , Proteínas Recombinantes/biosíntesis , Serpientes , Espectrofotometría UltravioletaRESUMEN
A gene which codes for the 66-residue polypeptide of kappa-bungarotoxin has been chemically synthesized by linking together 3 synthetic double-stranded oligonucleotides in a bacterial plasmid. The synthesis incorporated six unique silent restriction sites spaced throughout the gene for use in cassette mutagenesis. Direct expression of the kappa-bungarotoxin polypeptide by itself in Escherichia coli failed to result in a stable product. The toxin polypeptide was stabilized and expressed in E. coli as part of a fusion protein with rat intestinal fatty acid binding protein under control of the nalidixic acid inducible recA promoter. Two fusion protein constructs were prepared that differed only in the cleavage site between the fatty acid binding protein and the toxin polypeptide. One contained a factor Xa cleavage site, and the other, since the toxin itself is devoid of methionine, contained a methionyl residue that served as a cyanogen bromide cleavage site. The fusion proteins were isolated by ion-exchange chromatography and reverse-phase HPLC. The construct containing the factor Xa cleavage site could not be cleaved under nondenaturing conditions. On the other hand, kappa-bungarotoxin was efficiently cleaved from the methionyl fusion protein with CNBr. The toxin polypeptide was isolated by reverse-phase HPLC and ion-exchange chromatography and produced a complete and specific blockade of neuronal nicotinic acetylcholine receptors in chick ciliary ganglia which was indistinguishable from that produced by a comparable amount of venom-purified kappa-bungarotoxin.