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Protein Expr Purif. 2000 Jun;19(1):125-30.

Cloning, expression, and one-step purification of the minimal essential domain of the light chain of botulinum neurotoxin type A.

Author information

1
Biology and Biotechnology Research Program, Lawrence Livermore National Laboratory, Livermore, California 94551, USA. Kadkhodayan2@llnl.gov

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.

PMID:
10833399
DOI:
10.1006/prep.2000.1227
[Indexed for MEDLINE]

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