A recombinant scFv/streptavidin-binding peptide fusion protein for the quantitative determination of the scorpion venom neurotoxin AahI.
We created a construct encoding a peptide known to mimic the binding properties of biotin fused to the carboxy-terminus of a scFv fragment that binds a scorpion toxin (AahI). This fusion protein was produced in the periplasm of bacteria and purified to homogeneity by single-step affinity chromatography on streptavidin-agarose with a yield close to 1 mg/l. DNA sequencing, dot blot and mass spectrometric analyses demonstrated the integrity of the soluble immunoconjugate. Fusion to the streptavidin-binding peptide did not affect the ability of the scFv to recognize its antigen with a high affinity (Kd = 2.3 x 10(-10) M). Similarly, the streptavidin-binding property was not impaired in the fusion protein. Thus, the immunoconjugate was bifunctional and had a low molecular mass of 28 kDa. This enabled us to develop rapid and sensitive immunoassays for the specific detection of the toxin AahI accurately to 0.6 ng/ml, opening up new perspectives for the diagnosis of envenomations.
Aubrey
N
N
Muséum National d'Histoire Naturelle, LERAI, Paris, France.
Devaux
C
C
di Luccio
E
E
Goyffon
M
M
Rochat
H
H
Billiald
P
P
D000595:Amino Acid Sequence; D002352:Carrier Proteins / Q000737:chemistry*; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay; D008969:Molecular Sequence Data; D009498:Neurotoxins / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry; D012604:Scorpion Venoms / Q000737:chemistry*
D002352:Carrier Proteins; D009498:Neurotoxins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D012604:Scorpion Venoms; C452700:streptavidin-binding peptide; C065314:scorpion toxin I, Androctonus