PMID:10833399
| Citation |
Kadkhodayan, S, Knapp, MS, Schmidt, JJ, Fabes, SE, Rupp, B and Balhorn, R (2000) Cloning, expression, and one-step purification of the minimal essential domain of the light chain of botulinum neurotoxin type A. Protein Expr. Purif. 19:125-30 |
|---|---|
| Abstract |
A truncated but functional form of the botulinum neurotoxin A light chain (Tyr 9-Leu 415) has been cloned into the three bacterial expression vectors, pET 28, pET 30, and PGEX-2T, and produced as fusion proteins. This 406-amino-acid light chain was expressed with 1 six-histidine tag (LC-pET28), 2 six histidine tags and a S-tag (LC-pET30), or a six-histidine tag and a glutathione S-transferase tag (LC-pGEX-2T). The three fusion proteins have been overexpressed in Escherichia coli, purified in a soluble form, and tested for protease activity. All three recombinant proteins were found to have similar enzymatic activity, comparable to the light chain purified from the whole toxin. The LC-pET30 protein was the most soluble and stable of the three fusion proteins, and it could be purified using a one-step affinity chromatography protocol. The purified protein was determined to be 98% pure as assessed by SDS-polyacrylamide gel. This protein has been crystallized and initial X-ray data show that the crystals diffract to 1.8 A. |
| Links |
PubMed Online version:10.1006/prep.2000.1227 |
| Keywords |
Botulinum Toxins, Type A/chemistry; Botulinum Toxins, Type A/genetics; Botulinum Toxins, Type A/isolation & purification; Cloning, Molecular; Crystallography, X-Ray; Electrophoresis, Polyacrylamide Gel; Enzyme Stability; Escherichia coli/enzymology; Escherichia coli/genetics; Glutathione Transferase/chemistry; Histidine/chemistry; Membrane Proteins; Nerve Tissue Proteins/chemistry; Neuromuscular Agents/chemistry; Neuromuscular Agents/isolation & purification; Peptide Hydrolases/chemistry; Peptide Hydrolases/genetics; Peptide Hydrolases/isolation & purification; Protein Structure, Tertiary; Recombinant Fusion Proteins/chemistry; Recombinant Fusion Proteins/genetics; Recombinant Fusion Proteins/isolation & purification; Sequence Analysis, Protein; Synaptosomal-Associated Protein 25 |
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