1. Recombinant and Chemical Derivatives of Apamin. Implication of Post-Transcriptional C-Terminal Amidation of Apamin in Biological Activity
- Author
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Claude Granier, Daniel Baty, M.-L. Defendini, Martine Knibiehler, J. Van Rietschoten, Hervé Rochat, Christiane Devaux, and Kamel Mabrouk
- Subjects
Lysis ,Recombinant Fusion Proteins ,Molecular Sequence Data ,medicine.disease_cause ,Apamin ,complex mixtures ,Biochemistry ,law.invention ,Iodine Radioisotopes ,chemistry.chemical_compound ,law ,medicine ,Neurotoxin ,Amino Acid Sequence ,Escherichia coli ,Chromatography, High Pressure Liquid ,Base Sequence ,Chemistry ,Toxin ,Circular Dichroism ,Hydrolysis ,Antibodies, Monoclonal ,Biological activity ,Amides ,Fusion protein ,Colicin ,Recombinant DNA ,Protein Processing, Post-Translational - Abstract
The use of the colicin A lysis protein to direct the extracellular release of a fusion protein from Escherichia coli was investigated as an approach for the preparation of recombinant animal toxins. Apamin, a bee venom neurotoxin, was used as the model toxin. It is reticulated by two disulfide bridges and interacts with small conductance Ca(2+)-activated K+ channels. Substantial amounts of free recombinant apamin were obtained by CNBr cleavage of the fusion protein [col-(1-171)-apa] and HPLC purification. It was recognized by conformation-dependent monoclonal antibodies with a K0.5 value close to that for natural apamin, indicating that folding was correct. In toxicity and binding experiments, the recombinant apamin displayed low activity. The recombinant and natural molecules differed by the amidation of the C-terminal histidine residue. Previous structure/activity relationship studies do not implicate this C-terminal residue in activity but the role of its amidation was not investigated. An apamin analog with a non-amidated C-terminal residue was then chemically synthesized. The biological properties of both recombinant and chemical molecules were determined. Amidation of the C-terminal alpha-carboxyl of apamin appears to be essential for full expression of its biological activity.
- Published
- 1995