Escherichia coli heat-labile enterotoxin (LT) is composed of catalytic A and non-catalytic homo-pentameric B subunits and causes diarrheal disease in human and animals. In order to produce a nontoxic LT for vaccine and adjuvant development, two novel derivatives of LT were constructed by a site-directed mutagenesis of A subunit; Ser63 to Tyr63 in LTS63Y and Glu110, Glu112 were deleted in LT delta 110/112. The purified mutant LTs (mLTs) showed a similar molecular structural complex as AB5 to that of wild LT. In contrast to wild-type LT, mLTs failed to induce either elongation activity, ADP-ribosyltransferase activity, cAMP synthesis in CHO cells or fluid accumulation in mouse small intestine in vivo. Mice immunized with mLTs either intragastrically or intranasally elicited high titers of LT-specific serum and mucosal antibodies comparable to those induced by wild-type LT. These results indicate that substitution of Ser63 to Tyr63 or deletion of Glu110 and Glu112 eliminate the toxicity of LT without a change of AB5 conformation, and both mutants are immunogenic to LT itself. Therefore, both mLTs may be used to develop novel anti-diarrheal vaccines against enterotoxigenic E. coli.
Amino Acid Substitution ; Animal ; Bacterial Toxins/toxicity* ; Bacterial Toxins/metabolism ; Bacterial Toxins/immunology* ; Bacterial Toxins/genetics ; CHO Cells ; Cyclic AMP/metabolism ; Enterotoxins/toxicity* ; Enterotoxins/metabolism ; Enterotoxins/immunology* ; Enterotoxins/genetics ; Enzyme-Linked Immunosorbent Assay ; Escherichia coli*/metabolism ; Escherichia coli*/genetics ; Female ; Hamsters ; IgA, Secretory/blood ; Ileum/metabolism ; Immunity, Mucosal ; Mice ; Mice, Inbred BALB C ; Mutagenesis, Site-Directed ; NAD+ ADP-Ribosyltransferase/metabolism ; Recombinant Proteins/toxicity ; Recombinant Proteins/metabolism ; Recombinant Proteins/immunology ; Recombinant Proteins/chemistry

Heat-labile enterotoxin (LT) from Escherichia coli is a bacterial protein toxin with an AB5 hexamer structure. LT is a powerful mucosal adjuvant when co-administered with soluble antigens. However, its use in mucosal immunity is inconvenient because of its low yield and depolymerization during long-term storage under normal condition. In this study, we report an efficient expression system and optimized purification and storage strategy of LT. A gene encoding LT was cloned into the vector pET11c and transformed in E. coli BL21(DE3). By growing this strain on modified M9-CAA medium, LT was expressed efficiently. About 46mg/L LT could be purified from the supernatant of bacteria lysate. Using D(+)-Immobilized galactose column, LT could be purified at a wide pH range with various elution buffers. The optimized elution buffers are TEAN (pH 7.3) containing 0.3mol/L galactose and carbonate buffer (pH 10.4) containing 0.3mol/L galactose. After dried by freeze and placed in 4 degrees C, LT dissolved in TEAN (pH 7.3) and carbonate buffer (pH 10.4) were assayed by HPLC. The results indicated that the integrity of AB5 hexamer was kept well. LT could undergo long-term storage under this condition. This was proved to be an optimized strategy of LT storage. The results of GM1 binding assay and toxicity assay showed that the purified recombinant LT has normal biological character.
Animals ; Bacterial Toxins ; genetics ; isolation & purification ; metabolism ; pharmacology ; CHO Cells ; Cell Shape ; drug effects ; Chromatography, High Pressure Liquid ; Cricetinae ; Cricetulus ; Electrophoresis, Polyacrylamide Gel ; Enterotoxins ; genetics ; isolation & purification ; metabolism ; pharmacology ; Escherichia coli ; genetics ; metabolism ; Escherichia coli Proteins ; genetics ; isolation & purification ; metabolism ; pharmacology ; Female ; Genetic Vectors ; genetics ; Mice ; Mice, Inbred C57BL ; Toxicity Tests