Cloning, expression, and one-step purification of the minimal essential domain of the light chain of botulinum neurotoxin type A.
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.
Kadkhodayan
S
S
Biology and Biotechnology Research Program, Lawrence Livermore National Laboratory, Livermore, California 94551, USA. Kadkhodayan2@llnl.gov
Knapp
M S
MS
Schmidt
J J
JJ
Fabes
S E
SE
Rupp
B
B
Balhorn
R
R
D019274:Botulinum Toxins, Type A / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005982:Glutathione Transferase / Q000737:chemistry; D006639:Histidine / Q000737:chemistry; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins / Q000737:chemistry; D009465:Neuromuscular Agents / Q000737:chemistry / Q000302:isolation & purification*; D010447:Peptide Hydrolases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D020539:Sequence Analysis, Protein; D050825:Synaptosomal-Associated Protein 25
D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D009465:Neuromuscular Agents; D011993:Recombinant Fusion Proteins; D050825:Synaptosomal-Associated Protein 25; D006639:Histidine; D005982:Glutathione Transferase; D010447:Peptide Hydrolases; D019274:Botulinum Toxins, Type A