Hyperproduction of heat-stable enterotoxin (STA4) of Escherichia coli and analysis of the unusual electrophoretic behavior of reduced and alkylated forms of STAs.
The methanol-soluble heat-stable enterotoxin gene (estA4) of Escherichia coli (STA4) yielded 128-fold more toxin when expressed by a T7 RNA polymerase driven system than when driven by its own promoter. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of in vivo [35S]cysteine radiolabeled products of the cloned gene revealed an apparent molecular mass larger than that expected for a 19 amino acid polypeptide (mol. wt. 2049). Purified [125I]radiolabeled enterotoxin, STA1 (mol. wt. 1979) showed an Mr of 3800 when reduced, 2000 when reduced and carboxylated, and 14,500 when reduced and carboxyamidated. Similar changes after carboxyamidation were obtained with two different chemically synthesized STAs. These unusual electrophoretic mobilities were shown to be common to all STAs studied. Alkylation of the reduced STA species occurred only at the six cysteine residues of the toxin. Upon gel filtration the native, reduced, and reduced and alkylated forms of STAs eluted from the column in close agreement to the molecular weight expected from the known amino acid composition of the peptides.
Rasheed
J K
JK
Department of Microbiology, University of Texas Southwestern Medical Center, Dallas 75235.
Guzman-Verduzco
L M
LM
Kupersztoch
Y M
YM
D000478:Alkylation; D000595:Amino Acid Sequence; D001427:Bacterial Toxins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D003545:Cysteine / Q000378:metabolism; D004768:Enterotoxins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D005796:Genes; D005798:Genes, Bacterial; D007457:Iodine Radioisotopes; D010084:Oxidation-Reduction; D011401:Promoter Regions, Genetic; D013462:Sulfur Radioisotopes
D001427:Bacterial Toxins; D004768:Enterotoxins; D029968:Escherichia coli Proteins; D007457:Iodine Radioisotopes; D013462:Sulfur Radioisotopes; C034151:heat stable toxin (E coli); D003545:Cysteine