Expression, purification, and characterization of Clostridium botulinum type B light chain.
A full-length synthetic gene encoding the light chain of botulinum neurotoxin serotype B, approximately 50 kDa (BoNT/B LC), has been cloned into a bacterial expression vector pET24a+. BoNT/B LC was expressed in Escherichia coli BL21.DE3.pLysS and isolated from the soluble fraction. The resultant protein was purified to homogeneity by cation chromatography and was determined to be >98% pure as assessed by SDS-polyacrylamide gel stained with SilverXpress and analyzed by densitometry. Mass spectroscopic analysis indicated the protein to be 50.8 kDa, which equaled the theoretically expected mass. N-terminal sequencing of the purified protein showed the sequence corresponded to the known reported sequence. The recombinant BoNT/B light chain was found to be highly stable, catalytically active, and has been used to prepare antisera that neutralizes against BoNT/B challenge. Characterization of the protein including pH, temperature, and the stability of the protein in the presence or absence of zinc is described within. The influence of pH differences, buffer, and added zinc on secondary and tertiary structure of BoNT/B light chain was analyzed by circular dichroism and tryptophan fluorescence measurements. Optimal conditions for obtaining maximum metalloprotease activity and stabilizing the protein for long term storage were determined. We further analyzed the thermal denaturation of BoNT/B LC as a function of temperature to probe the pH and added zinc effects on light chain stability. The synthetic BoNT/B LC has been found to be highly active on its substrate (vesicle associated membrane protein-2) and, therefore, can serve as a useful reagent for BoNT/B research.
Gilsdorf
Janice
J
Integrated Toxicology Division, U.S. Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Frederick, MD 21702-5011, USA.
Gul
Nizamettin
N
Smith
Leonard A
LA
D000818:Animals; D001905:Botulinum Toxins / Q000096:biosynthesis* / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification*; D019274:Botulinum Toxins, Type A; D004926:Escherichia coli; D006863:Hydrogen-Ion Concentration; D051379:Mice; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification*; D015032:Zinc / Q000737:chemistry
D011994:Recombinant Proteins; C096323:rimabotulinumtoxinB; D001905:Botulinum Toxins; D019274:Botulinum Toxins, Type A; D015032:Zinc