Cloning, expression, purification, and characterization of biologically active recombinant hemagglutinin-33, type A botulinum neurotoxin associated protein

• Published in: The Protein Journal 2007-01, Vol.26 (1), p.29-37
• Main Authors: Zhou, Yu, Paturi, Sowmya, Lindo, Paul, Shoesmith, Suzanne M, Singh, Bal Ram
• Format: Article
• Language: English
• Subjects: Aqueous solutions; Bacteria; Bacterial Proteins - chemistry; Bacterial Proteins - isolation & purification; Bacterial Proteins - pharmacology; Biological activity; Biosafety; Botulinum toxin; Botulinum Toxins - chemistry; Botulinum Toxins - isolation & purification; Botulinum Toxins - pharmacology; Botulism; Chromatography, Affinity; Circular Dichroism; Cloning; Cloning, Molecular - methods; Clostridium botulinum - chemistry; Clostridium botulinum - genetics; Crystallography, X-Ray; Denaturation; Dichroism; E coli; Electrophoresis, Polyacrylamide Gel; Escherichia coli - chemistry; Escherichia coli - genetics; Gene Expression; Genes; Hemagglutination; Hemagglutination Tests; Hemagglutinins; Models, Molecular; Molecular Weight; Neurotoxins; Protein Structure, Secondary; Proteins; Proteolysis; Recombinant Proteins - biosynthesis; Recombinant Proteins - chemistry; Recombinant Proteins - isolation & purification; Toxic substances; Toxins; Trypsin; Trypsin - metabolism
• ISSN: 1572-3887; 1875-8355; 1573-4943
• DOI: 10.1007/s10930-006-9041-4

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.