Expression and purification of the light chain of botulinum neurotoxin A: a single mutation abolishes its cleavage of SNAP-25 and neurotoxicity after reconstitution with the heavy chain
| Autor: | Roger K. Aoki, J.O. Dolly, Liqing Zhou, A. De Paiva, Dandan Liu |
|---|---|
| Rok vydání: | 1995 |
| Předmět: |
Botulinum Toxins
Monosaccharide Transport Proteins Recombinant Fusion Proteins Mutant Molecular Sequence Data Neuromuscular transmission Gene Expression Immunoglobulin light chain Biochemistry Polymerase Chain Reaction Chromatography Affinity Maltose-Binding Proteins law.invention Maltose-binding protein Mice Structure-Activity Relationship Affinity chromatography law Endopeptidases Escherichia coli Neurotoxin Animals Amino Acid Sequence biology Base Sequence Chemistry Escherichia coli Proteins Botulism Molecular biology Fusion protein Peptide Fragments Zinc Mutagenesis Recombinant DNA biology.protein ATP-Binding Cassette Transporters Neuromuscular Blocking Agents Carrier Proteins |
| Zdroj: | Biochemistry. 34(46) |
| ISSN: | 0006-2960 |
| Popis: | Botulinum neurotoxin type A (BoNT/A) selectively and irreversibly inhibits acetylcholine release from peripheral nerve endings. While the toxin's heavy (H) chain contributes to neuronal binding and internalization, its light (L) chain is a Zn(2+)-dependent endoprotease that intracellularly cleaves synaptosomal-associated protein of M(r) = 25 kDa (SNAP-25). For research and clinical exploitation of this uniquely-acting neurotoxin, recombinant wild-type L chain was produced together with a mutant in which His227 in the Zn(2+)-binding motif was substituted by Tyr. The PCR-amplified wild-type and mutant L chain genes were cloned, fused to the gene for maltose-binding protein, and expressed at high levels in Escherichia coli. The soluble fusion proteins were purified using amylose affinity chromatography, and, after factor Xa cleavage, the free L chains were isolated. The wild-type was shown to proteolyze SNAP-25 at a rate approaching that of the native chain while the mutant was inactive. Reconstitution of the pure wild-type L chain with native homogeneous H chain yielded a disulfide-linked dichain form that inhibited neuromuscular transmission in vitro and produced the symptoms of botulism in vivo. After reconstitution with the H chain, the Tyr227 mutant L chain failed to show any neuroparalytic activity in either of these assays. This methodology allows, for the first time, routine preparation of recombinant forms of the L chain that are needed to decipher the molecular details of its interaction with substrate and, thereby, assist the design of effective inhibitors.(ABSTRACT TRUNCATED AT 250 WORDS) |
| Databáze: | OpenAIRE |
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