Functional expression and site-directed mutagenesis of a synthetic gene for alpha-bungarotoxin

• Published in: The Journal of biological chemistry Vol. 269; no. 15; pp. 11178 - 11185
• Main Authors: ROSENTHAL, J. A, HSU, S. H, SCHNEIDER, D, GENTILE, L. N, MESSIER, N. J, VASLET, C. A, HAWROT, E
• Format: Journal Article
• Language: English
• Published: Bethesda, MD American Society for Biochemistry and Molecular Biology 15.04.1994
• Subjects: Alanine; Amino Acid Sequence; Analytical, structural and metabolic biochemistry; Animals; Aspartic Acid; Base Sequence; Biological and medical sciences; Bungarotoxins - biosynthesis; Bungarotoxins - isolation & purification; Bungarotoxins - metabolism; Bungarus multicinctus; Cell Membrane - metabolism; Chromatography, DEAE-Cellulose; Cloning, Molecular; Electric Organ - metabolism; Escherichia coli; Factor Xa - metabolism; Fundamental and applied biological sciences. Psychology; Gene Expression; Genes, Synthetic; Miscellaneous; Models, Molecular; Molecular Sequence Data; Mutagenesis, Site-Directed; Oligodeoxyribonucleotides - chemical synthesis; Point Mutation; Protein Folding; Protein Structure, Secondary; Proteins; Recombinant Fusion Proteins - biosynthesis; Recombinant Fusion Proteins - isolation & purification; Recombinant Proteins - biosynthesis; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; Snakes; Torpedo - metabolism; Ligand binding; Cholinergic receptor; Recombinant protein; Invertebrata; Neurotoxin; Site directed mutagenesis
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(19)78107-1

In order to explore the structure-function relationships of the curare mimetic alpha-neurotoxins we have constructed and cloned a synthetic gene for Bungarus multicinctus alpha-bungarotoxin which is expressed in Escherichia coli. The recombinant alpha-bungarotoxin is expressed as a fusion protein with alpha-bungarotoxin linked to the COOH-terminal end of the T7 Gene 9-encoded coat protein. After treatment of the fusion protein with Factor Xa protease, a recombinant alpha-bungarotoxin is released that co-migrates with authentic alpha-bungarotoxin upon reverse-phase high performance liquid chromatography and ion-exchange chromatography. Final yields of active recombinant alpha-bungarotoxin were about 0.4 mg/liter of starting bacterial culture. The recombinant alpha-bungarotoxin contains 10 additional residues linked to the NH2-terminal Ile of the alpha-bungarotoxin sequence due apparently to the inaccessibility of the engineered cleavage site to Factor Xa. Nevertheless, the recombinant alpha-bungarotoxin is capable of binding to the nicotinic acetylcholine receptor with an apparent affinity that is only decreased approximately 1.7-fold from that of authentic alpha-bungarotoxin. Alanine substitution of a residue, Asp30, highly conserved among alpha-neurotoxins and previously suggested to play a key role in receptor recognition, resulted in a recombinant alpha-bungarotoxin whose receptor binding activity is indistinguishable from authentic alpha-bungarotoxin.