Functional expression and site-directed mutagenesis of a synthetic gene for alpha-bungarotoxin.
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.
Rosenthal
J A
JA
Section of Molecular and Biochemical Pharmacology, Brown University, Providence, Rhode Island 02912.
Hsu
S H
SH
Schneider
D
D
Gentile
L N
LN
Messier
N J
NJ
Vaslet
C A
CA
Hawrot
E
E
D000409:Alanine; D000595:Amino Acid Sequence; D000818:Animals; D001224:Aspartic Acid; D001483:Base Sequence; D002038:Bungarotoxins / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D002848:Chromatography, DEAE-Cellulose; D003001:Cloning, Molecular; D004557:Electric Organ / Q000378:metabolism; D004926:Escherichia coli; D015951:Factor Xa / Q000378:metabolism; D015870:Gene Expression; D005813:Genes, Synthetic; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis; D017354:Point Mutation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D012911:Snakes; D014101:Torpedo / Q000378:metabolism
D002038:Bungarotoxins; D009838:Oligodeoxyribonucleotides; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D001224:Aspartic Acid; D015951:Factor Xa; D000409:Alanine