Insertion of a disulfide-containing neurotoxin into E. coli alkaline phosphatase: the hybrid retains both biological activities.
We have inserted a disulfide-containing snake neurotoxin into the N-terminal end of Escherichia coli alkaline phosphatase, between residues +6 and +7 of the mature enzyme. For this purpose, we have designed a cloning and expression vector which allows insertion of foreign DNA between the corresponding codons, and visual selection of the desired recombinant clones upon recovery of phosphatase activity. The hybrid protein is exported to the bacterial periplasm, the alkaline phosphatase signal peptide is correctly processed, and both domains are functionally conformed. The phosphatase domain displays catalytic activity, and the inserted toxin is able to bind to its biological target, the nicotinic acetylcholine receptor. The hybrid molecule is remarkably stable and resistant to proteolysis. Crude periplasmic extract containing the hybrid can be used as a tracer-containing reagent in competitive enzymo-immuno and enzymo-receptor assays. We propose to use the system described in this paper for fast preparation of properly folded disulfide-containing enzymatic probes.
Gillet
D
D
Département d'Ingénierie et d'Etude des Protéines, CEA Saclay, Gif sur Yvette, France.
Ducancel
F
F
Pradel
E
E
Léonetti
M
M
Ménez
A
A
Boulain
J C
JC
D000469:Alkaline Phosphatase / Q000737:chemistry* / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D004220:Disulfides; D004797:Enzyme-Linked Immunosorbent Assay; D004870:Erabutoxins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009498:Neurotoxins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010957:Plasmids; D011233:Precipitin Tests; D015202:Protein Engineering; D011978:Receptors, Nicotinic / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism
D004220:Disulfides; D009498:Neurotoxins; D011978:Receptors, Nicotinic; D011993:Recombinant Fusion Proteins; C038908:erabutoxin A; D004870:Erabutoxins; D000469:Alkaline Phosphatase