[Expression of heat-labile enterotoxin and the strategy of purification and storage].
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.
Feng
Qiang
Q
College of Bioengineering, Chongqing University, Chongqing 400044, China.
Cai
Shao-Xi
SX
Yang
Jun
J
Luo
Ping
P
Zhang
Wei-Jun
WJ
Zou
Quan-Ming
QM
D000818:Animals; D001427:Bacterial Toxins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism* / Q000494:pharmacology; D016466:CHO Cells; D048430:Cell Shape / Q000187:drug effects; D002851:Chromatography, High Pressure Liquid; D006224:Cricetinae; D003412:Cricetulus; D004591:Electrophoresis, Polyacrylamide Gel; D004768:Enterotoxins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism* / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism* / Q000494:pharmacology; D005260:Female; D005822:Genetic Vectors / Q000235:genetics; D051379:Mice; D008810:Mice, Inbred C57BL; D018675:Toxicity Tests
D001427:Bacterial Toxins; D004768:Enterotoxins; D029968:Escherichia coli Proteins; C027538:heat-labile enterotoxin, E coli