title: Cloning, expression, purification, and characterization of biologically active recombinant hemagglutinin-33, type A botulinum neurotoxin associated protein.
abstract: Botulinum neurotoxin type A, the most toxic substance known to mankind, is produced by Clostridiurn botulinum type A as a complex with a group of neurotoxin-associated proteins (NAPs) through polycistronic expression of a clustered group of genes. Hemagglutinin-33 (Hn-33) is a 33 kDa subcomponent of NAPs, which is resistant to protease digestion, a feature likely to be involved in the protection of the botulinum neurotoxin from proteolysis. In order to fully understand the function of Hn-33, large amounts of Hn-33 will be needed without dealing with biosafety risks to grow large cultures of C. botulinum. There are difficulties to clone the genes with the high A + T contents produced by C. botulinum. We report here for the first time using the Gateway technology to clone functional Hn-33 that has been expressed in E. coli. The yield of the recombinant Hn-33 was about 12 mg per liter of E. coli culture. The recombinant Hn-33 folds well in aqueous solution as shown with circular dichroism spectra, resists temperature-denaturation, is totally resistant to trypsin proteolysis despite the presence of cleavage sites on the molecular surface, and maintains its biological activities comparable to the native Hn-33 hemagglutination.
authors.affiliation: Department of Chemistry and Biochemistry, University of Massachusetts Dartmouth, 285 Old Westport Road, North Dartmouth, MA, 02747, USA.
authors.forename: Yu
authors.initials: Y
authors.lastname: Zhou
authors.forename: Sowmya
authors.initials: S
authors.lastname: Paturi
authors.forename: Paul
authors.initials: P
authors.lastname: Lindo
authors.forename: Suzanne M
authors.initials: SM
authors.lastname: Shoesmith
authors.forename: Bal Ram
authors.initials: BR
authors.lastname: Singh
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000302:isolation & purification* / Q000494:pharmacology; D001905:Botulinum Toxins / Q000737:chemistry* / Q000302:isolation & purification* / Q000494:pharmacology; D002846:Chromatography, Affinity; D002942:Circular Dichroism; D003001:Cloning, Molecular / Q000379:methods; D003014:Clostridium botulinum / Q000737:chemistry* / Q000235:genetics; D018360:Crystallography, X-Ray; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics; D015870:Gene Expression; D006386:Hemagglutination Tests; D008958:Models, Molecular; D008970:Molecular Weight; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D014357:Trypsin / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; C118973:hemagglutinin 33 protein, Clostridium botulinum; D014357:Trypsin; D001905:Botulinum Toxins