A recombinant scFv/streptavidin-binding peptide fusion protein for the quantitative determination of the scorpion venom neurotoxin AahI.
Biol Chem
; 382(11): 1621-8, 2001 Nov.
Article
em En
| MEDLINE
| ID: mdl-11767952
ABSTRACT
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.
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Base de dados:
MEDLINE
Assunto principal:
Venenos de Escorpião
/
Proteínas de Transporte
/
Neurotoxinas
Idioma:
En
Ano de publicação:
2001
Tipo de documento:
Article