title: Direct expression in E. coli of a functionally active protein A--snake toxin fusion protein.
abstract: We constructed a recombinant expression plasmid encoding a protein A--neurotoxin fusion protein. The fused toxin is directly expressed in the periplasmic space of Escherichia coli and can be purified in the milligram range by a single immuno-affinity step. The LD50 values of the fused toxin and native toxin are 130 and 20 nmol/kg mouse respectively. The Kd values characterizing their binding to the nicotinic acetylcholine receptor (AcChoR) are respectively 4.8 +/- 0.8 and 0.07 +/- 0.03 nM. In contrast, the fused and native toxins are equally well recognized by a toxin-specific monoclonal antibody which recognizes the AcChoR binding site. The lower toxicity of the fused toxin might result, therefore, from a steric hindrance, due to the presence of the bulky protein A moiety (mol. wt = 31 kd) rather than to a direct alteration of the 'toxic' site. The fused toxin is more immunogenic than native toxin, since 1 nmol of hybrid toxin and 14 nmol of native toxin give rise to comparable titers of antitoxin antibodies which, furthermore, are equally potent at neutralizing neurotoxicity. The work described in this paper shows that the use of fused toxins may be of paramount importance for future development of serotherapy against envenomation by snake bites.
authors.affiliation: Département de Biologie, Laboratoire d'Ingenierie des Protéines, C.E.N., Gif/Yvette, France.
authors.forename: F
authors.initials: F
authors.lastname: Ducancel
authors.forename: J C
authors.initials: JC
authors.lastname: Boulain
authors.forename: O
authors.initials: O
authors.lastname: Trémeau
authors.forename: A
authors.initials: A
authors.lastname: Ménez
mesh_terms: D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D005260:Female; D015870:Gene Expression; D007928:Lethal Dose 50; D009498:Neurotoxins / Q000378:metabolism*; D010957:Plasmids; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000378:metabolism* / Q000633:toxicity; D012910:Snake Venoms / Q000378:metabolism*; D013205:Staphylococcal Protein A / Q000378:metabolism*
chemical_list: D009498:Neurotoxins; D011993:Recombinant Fusion Proteins; D012910:Snake Venoms; D013205:Staphylococcal Protein A