title: High-level expression, purification, and characterization of recombinant type A botulinum neurotoxin light chain.
abstract: Botulinum neurotoxin light chain (BoNT LC, 50 kDa) is responsible for the zinc endopeptidase activity specific for proteins of neuroexocytosis apparatus. We describe the expression of recombinant type A BoNT LC in Escherichia coli as well as the purification and characterization of the recombinant protein. A high level of expression of BoNT/A LC was obtained by an extended postinduction time of 15 h at 30 degrees C. Recombinant BoNT/A LC was isolated from an Ni(2+) column. Due to its high pI ( approximately 8.7), purification was achieved by a single step of passing the protein through anion-exchange chromatography at pH 8.0 without the need of elution. The purified recombinant BoNT/A LC retained proteolytic activity and had a secondary structure similar to that of native LC determined by CD measurement.
authors.affiliation: Department of Chemistry and Biochemistry, University of Massachusetts Dartmouth, Dartmouth, Massachusetts 02747, USA.
authors.forename: L
authors.initials: L
authors.lastname: Li
authors.forename: B R
authors.initials: BR
authors.lastname: Singh
mesh_terms: D019274:Botulinum Toxins, Type A / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D002852:Chromatography, Ion Exchange; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D007525:Isoelectric Focusing; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification
chemical_list: D011994:Recombinant Proteins; D010450:Endopeptidases; D019274:Botulinum Toxins, Type A