TY - JOUR
T1 - Expression and characterization of the cloned Salmonella typhimurium enterotoxin
AU - Prasad, Rajendra
AU - Chopra, Ashok K.
AU - Chary, Parvathi
AU - Peterson, Johnny W.
N1 - Funding Information:
This work was supported by research grant R01 18401-09 from the National Institutes of Health . The help of J . Cantu in conducting tissue culture work is greatly appreciated .
PY - 1992/8
Y1 - 1992/8
N2 - Earlier, our laboratory reported the cloning of a chromosomally encoded cholera toxin (CT)-like enterotoxin gene from Salmonella typhimurium Q1 into pBR322. Cell lysates from the plasmid clone pC1, containing a 4.8 kb EcoR1 DNA fragment from Salmonella, caused elongation of Chinese hamster ovary (CHO) cells and this biologic activity was neutralized by anti-CT. However, this cloned gene product did not elicit fluid secretion in the rabbit intestinal loop (RIL) model, because of poor expression. We report here, subcloning of a 4.8 kb EcoRI and a 2.7 kb HindIII EcoRI fragment into a high expression T7 RNA polymerase/promoter system. Cell lysates from these clones elicited fluid secretion in the RIL model, caused firm induration in rabbit skin and elongated CHO cells. These biological activities were neutralized by anti-CT. SDS-PAGE and subsequent fluorographic analysis of Escherichia coli, harboring recombinant plasmids in a T7 expression system, revealed the presence of two prominent 35S-labeled polypeptides of 25 and 12 kDa, which were immunoprecipitated with anti-CT. The enterotoxin appeared to be 125 kDa in size, based on chromatography on a P-300 column, had a pl of 6.6 to 6.8, and was heat-labile (60 °C/5 min). Unlike cloned CT and heat-labile enterotoxin (LT-I), which were localized in the periplasm, the Salmonella enterotoxin was cytoplasmic in nature.
AB - Earlier, our laboratory reported the cloning of a chromosomally encoded cholera toxin (CT)-like enterotoxin gene from Salmonella typhimurium Q1 into pBR322. Cell lysates from the plasmid clone pC1, containing a 4.8 kb EcoR1 DNA fragment from Salmonella, caused elongation of Chinese hamster ovary (CHO) cells and this biologic activity was neutralized by anti-CT. However, this cloned gene product did not elicit fluid secretion in the rabbit intestinal loop (RIL) model, because of poor expression. We report here, subcloning of a 4.8 kb EcoRI and a 2.7 kb HindIII EcoRI fragment into a high expression T7 RNA polymerase/promoter system. Cell lysates from these clones elicited fluid secretion in the RIL model, caused firm induration in rabbit skin and elongated CHO cells. These biological activities were neutralized by anti-CT. SDS-PAGE and subsequent fluorographic analysis of Escherichia coli, harboring recombinant plasmids in a T7 expression system, revealed the presence of two prominent 35S-labeled polypeptides of 25 and 12 kDa, which were immunoprecipitated with anti-CT. The enterotoxin appeared to be 125 kDa in size, based on chromatography on a P-300 column, had a pl of 6.6 to 6.8, and was heat-labile (60 °C/5 min). Unlike cloned CT and heat-labile enterotoxin (LT-I), which were localized in the periplasm, the Salmonella enterotoxin was cytoplasmic in nature.
KW - Chinese hamster ovary (CHO) cells
KW - G-ganglioside ELISA
KW - Salmonella enterotoxin (stn)
KW - T7 RNA polymerase/promoter
KW - cholera toxin
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U2 - 10.1016/0882-4010(92)90071-U
DO - 10.1016/0882-4010(92)90071-U
M3 - Article
C2 - 1453924
AN - SCOPUS:0026906892
SN - 0882-4010
VL - 13
SP - 109
EP - 121
JO - Microbial Pathogenesis
JF - Microbial Pathogenesis
IS - 2
ER -