High level expression of the light chain of botulinum neurotoxin serotype C1 and an efficient HPLC assay to monitor its proteolytic activity.
Botulinum neurotoxins (serotypes BoNT/A-BoNT/G) induce botulism, a disease leading to flaccid paralysis. These serotypes are highly specific in their proteolytic cleavage of SNAP-25 (synaptosomal-associated protein of 25kDa), VAMP (vesicle associated membrane protein) or syntaxin. The catalytic domain (light chain, LC) of the neurotoxin has a Zn(2+) dependent endopeptidase activity. In order to design drugs and inhibitors against these toxins, high level overexpression and characterization of LC of BoNTs along with the development of assays to monitor their proteolytic activity becomes important. Using the auto-induction method, we attained a high level expression of BoNT/C1(1-430) yielding more than 30mg protein per 500ml culture. We also developed an efficient assay to measure the activity of serotype C1 based on a HPLC method. SNAP-25 with varying peptide length has been reported in literature as substrates for BoNT/C1 proteolysis signifying the importance of remote exosites in BoNT/C1 required for activity. Here, we show that a 17-mer peptide corresponding to residues 187-203 of SNAP-25, which has earlier been shown to be a substrate for BoNT/A, can be used as a substrate for quantifying the activity of BoNT/C1(1-430). There was no pH dependence for the proteolysis, however the presence of dithiothreitol is essential for the reaction. Although the 17-mer substrate bound 110-fold less tightly to BoNT/C1(1-430) than SNAP-25, the optimal assay conditions facilitated an increase in the catalytic efficiency of the enzyme by about 5-fold.
Rawat
Richa
R
Biology Department, Brookhaven National Laboratory, Upton, NY 11973, USA.
Ashraf Ahmed
S
S
Swaminathan
Subramanyam
S
D001483:Base Sequence; D001905:Botulinum Toxins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000494:pharmacology; D020134:Catalytic Domain; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D002942:Circular Dichroism; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D013050:Spectrometry, Fluorescence
D017931:DNA Primers; D001905:Botulinum Toxins; C096324:botulinum toxin type C