High-level expression and secretion of a lysine-containing analog of Escherichia coli heat-stable enterotoxin.
The mechanism of action of the heat-stable enterotoxin STa secreted from enterotoxigenic forms of Escherichia coli has remained elusive, in part due to a tedious, low-yield purification procedure. We report here a method for obtaining large amounts of a biologically active lysine-containing analog of STa. Initial attempts to express the toxin using an expression vector that did not encode a signal sequence resulted in no biologically active material being recovered either from lysed cells or as a secretory product. However, use of the secretion vector pJAL36, which contains the STII enterotoxin signal sequence, allowed large amounts of an STa derivative containing the additional sequence Ser-Thr-Lys at the amino terminus of the mature enterotoxin to be readily purified from culture supernatants. This enterotoxin analog, known as KSTa-1, was equal in biological and receptor binding activity to the native toxin STa. The lysine residue present in KSTa-1 promises to be useful as a reactive amino acid that is readily derivatized to allow coupling of the enterotoxin to supports for affinity chromatography and antigenic conjugates. Additionally, the insertion of the lysine residue carboxy terminal to the Ser-Thr sequence adds a reversible "handle" to the toxin sequence in that the Ser-Thr-Lys segment can be removed by treatment with trypsin, releasing the native form of STa.
Greenberg
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Department of Medicine, University of Kentucky, Lexington 40536.
Ping
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Biek
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Mann
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D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001427:Bacterial Toxins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D004768:Enterotoxins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D015870:Gene Expression; D005798:Genes, Bacterial; D005822:Genetic Vectors; D008239:Lysine / Q000737:chemistry; D008969:Molecular Sequence Data; D021382:Protein Sorting Signals / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics
D000596:Amino Acids; D001427:Bacterial Toxins; D004269:DNA, Bacterial; D004768:Enterotoxins; D029968:Escherichia coli Proteins; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; C034151:heat stable toxin (E coli); D008239:Lysine