Synthesis and expression in Escherichia coli of a gene for kappa-bungarotoxin.
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.
Fiordalisi
J J
JJ
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110.
Fetter
C H
CH
TenHarmsel
A
A
Gigowski
R
R
Chiappinelli
V A
VA
Grant
G A
GA
D000595:Amino Acid Sequence; D000818:Animals; D002038:Bungarotoxins / Q000096:biosynthesis / Q000235:genetics*; D002645:Chickens; D004926:Escherichia coli / Q000235:genetics*; D005813:Genes, Synthetic; D005818:Genetic Engineering; D005822:Genetic Vectors; D006868:Hydrolysis; D008969:Molecular Sequence Data; D010957:Plasmids; D011817:Rabbits; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification
D002038:Bungarotoxins; D011993:Recombinant Fusion Proteins