A Recombinant scFv/Streptavidin-Binding Peptide Fusion Protein for the Quantitative Determination of the Scorpion Venom Neurotoxin AahI
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Nicolas Aubrey
Abstract
We created a construct encoding a peptide known to mimic the binding properties of biotin fused to the carboxyterminus 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 singlestep affinity chromatography on streptavidinagarose 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 streptavidinbinding peptide did not affect the ability of the scFv to recognize its antigen with a high affinity (Kd = 2.3x 1010M). Similarly, the streptavidinbinding 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.
Copyright © 2001 by Walter de Gruyter GmbH & Co. KG
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Artikel in diesem Heft
- An Overview of Endosymbiotic Models for the Origins of Eukaryotes, Their ATP-Producing Organelles (Mitochondria and Hydrogenosomes), and Their Heterotrophic Lifestyle
- A Microsomal Ecdysone-Binding Cytochrome P450 from the Insect Locusta migratoria Purified by Sequential Use of Type-II and Type-I Ligands
- The Large Cytoplasmic Loop of the Glucose Transporter GLUT1 Is an Essential Structural Element for Function
- Visual Representation by Atomic Force Microscopy (AFM) of Tomato Spotted Wilt Virus Ribonucleoproteins
- Elimination of Hydrocortisone from the Medium Enables Tissue Plasminogen Activator Gene Expression by Normal and Immortalized Nonmalignant Human Epithelial Cells
- Molecular Cloning and Biochemical Characterisation of Proteases from Staphylococcus epidermidis
- Splice Variants of Human Cathepsin L mRNA Show Different Expression Rates
- Osmolytes as Modulators of Conformational Changes in Serpins
- Protective Activity of Aromatic Amines and Imines against Oxidative Nerve Cell Death
- Nitric Oxide Generation in Aqueous Solutions of Cigarette Smoke and Approaches to Its Origin
- A Recombinant scFv/Streptavidin-Binding Peptide Fusion Protein for the Quantitative Determination of the Scorpion Venom Neurotoxin AahI
