Recombinant cholera toxin B subunit in Escherichia coli: high-level secretion, purification, and characterization.
The gene coding for cholera toxin subunit B (CT-B) was fused to a modified ompA signal sequence and subsequently cloned into a high expression vector based on the regulatory signals of the arabinose operon of Salmonella typhimurium. Upon induction of gene expression in Escherichia coli, a product of the expected size for CT-B monomer was detected at a level of approximately 60% of total periplasmic protein. At pilot scale, batch cultivation in a 20-liter bioreactor allowed a production level of 1 g/liter of recombinant CT-B (rCT-B), the majority of which was released into the culture medium. The latter phenomenon was dependent on the medium selected for cultivation. A simple and inexpensive purification scheme was developed which enabled the recovery of 81% of rCT-B from the culture supernatant. Comparing amino acid composition, amino-terminal sequence, mass spectrum, pentamerisation, and GM1-binding, rCT-B is indistinguishable from natural CT-B produced by Vibrio cholerae. This rCT-B overproducing E. coli strain represents an interesting alternative to overexpressing systems developed in V. cholerae.
Slos
P
P
Department of Bacterial Vectors, TRANSGENE S. A., Strasbourg, France.
Speck
D
D
Accart
N
N
Kolbe
H V
HV
Schubnel
D
D
Bouchon
B
B
Bischoff
R
R
Kieny
M P
MP
D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D002772:Cholera Toxin / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D002850:Chromatography, Gel; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D021382:Protein Sorting Signals / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D014734:Vibrio cholerae / Q000235:genetics
D001425:Bacterial Outer Membrane Proteins; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D002772:Cholera Toxin