Cloning, high level expression, purification, and crystallization of the full length Clostridium botulinum neurotoxin type E light chain.
The catalytic activity of the highly potent botulinum neurotoxins are confined to their N-terminal light chains ( approximately 50kDa). A full-length light chain for the type E neurotoxin with a C-terminal 6x His-tag, BoNT/E-LC, has been cloned in a pET-9c vector and over-expressed in BL21 (DE3) cells. BoNT/E-LC was purified to homogeneity by affinity chromatography on Ni-NTA agarose followed by exclusion chromatography using a Superdex-75 sizing column. The purified protein has very good solubility and can be stored stably at -20 degrees C; however, it seems to undergo auto-proteolysis when stored at temperature #10878;4-10 degrees C. BoNT/E-LC is active on its natural substrate, the synaptosomal associated 25kDa protein, SNAP-25, indicating that it retains a native-like conformation and therefore can be considered as a useful tool in studying the structure/function of the catalytic light chain. Recombinant BoNT/E-LC has been crystallized under five different conditions and at various pHs. Crystals diffract to better than 2.1A.
Agarwal
Rakhi
R
Biology Department, Brookhaven National Laboratory, Upton, NY 11973, USA.
Eswaramoorthy
Subramaniam
S
Kumaran
Desigan
D
Dunn
John J
JJ
Swaminathan
Subramanyam
S
D001905:Botulinum Toxins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D002384:Catalysis; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D003014:Clostridium botulinum / Q000201:enzymology / Q000235:genetics; D003460:Crystallization; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics; D006639:Histidine / Q000235:genetics; D007700:Kinetics; D008565:Membrane Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D021122:Protein Subunits / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D012995:Solubility; D050825:Synaptosomal-Associated Protein 25; D013696:Temperature; D013997:Time Factors
D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; D050825:Synaptosomal-Associated Protein 25; D006639:Histidine; D001905:Botulinum Toxins; C096326:botulinum toxin type E