Efficient production of Clostridium botulinum exotoxin C3 in bacteria: a screening method to optimize production yields.
Clostridium botulinum exoenzyme C3 is responsible for the inactivation of members of the Rho GTPase family that are implicated in actin-cytoskeleton reorganization. This property has been extensively used in the field to investigate the functionality of the Rho GTPases. However, systematic analysis of Rho GTPase functions requires large amounts of such inhibitors and consequently an optimization of the production yield of these proteins. Bacterial production of soluble proteins often requires a refolding step that noticeably affects the production yields and necessitates additional experiments to verify functional activity. This is particularly true for TAT-C3, the production yields of which are generally low. In this report, we describe a rapid and efficient method for the production of soluble C3 exoenzyme developed by screening a collection of bacterial strains. The recombinant C3 protein was fused to the TAT protein-transduction domain from HIV, to allow protein delivery into cells, and to a hexahistidine tag, that permitted purification by Nickel affinity chromatography. We have demonstrated the production of large amounts of soluble and functional protein using the bacterial strain AD494 (DE3)pLysS. This rapid and efficient method for the production of soluble C3 exoenzyme could also be useful for the production of other proteins with solubility problems.
Gadea
Gilles
G
Centre de Recherche en Biochimie Macromoléculaire, CNRS FRE 2593, IFR 122, 1919 route de Mende, F-34293 Montpellier cedex 5, France.
Boublik
Yvan
Y
Delga
Stephanie
S
Roux
Pierre
P
D036002:ADP Ribose Transferases / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification; D001905:Botulinum Toxins / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification; D002942:Circular Dichroism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015202:Protein Engineering / Q000379:methods
D036002:ADP Ribose Transferases; C063007:exoenzyme C3, Clostridium botulinum; D001905:Botulinum Toxins