Synthesis of Escherichia coli heat-stable enterotoxin STp as a pre-pro form and role of the pro sequence in secretion.
Escherichia coli heat-stable enterotoxin STp is presumed from its DNA sequence to be synthesized in vivo as a 72-amino-acid residue precursor that is cleaved to generate mature STp consisting of the 18 carboxy-terminal amino acid residues. There are two methionine residues in the inferred STp sequence in addition to the methionine residue at position 1. In order to confirm production of the STp 72-amino-acid residue precursor, we substituted the additional methionine residues by oligonucleotide-directed site-specific mutagenesis. Since these substitutions did not cause a significant change in STp production, it can be concluded that STp is normally synthesized as the 72-amino-acid residue precursor. The length of the STp precursor indicated the existence of a pro sequence between the signal peptide and the mature protein. In order to identify the pro sequence and determine its role in protein secretion, deletion and fusion proteins were made. A deletion mutant in which the gene fragment encoding amino acid residues 22 to 53 of STp was removed was made. STp activity was found in the culture supernatant of cells. Amino acid sequence analysis of the purified STp deletion mutant revealed that the pro sequence encompasses amino acid residues 20 to 54. A hybrid protein consisting of STp amino acids 1 to 53 fused in frame from residue 53 to nuclease A was not secreted into the culture supernatant. These results indicate that the pro sequence does not function to guide periplasmic protein into the extracellular milieu.
Okamoto
K
K
Department of Biochemistry, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Japan.
Takahara
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001427:Bacterial Toxins / Q000235:genetics* / Q000302:isolation & purification / Q000633:toxicity; D001483:Base Sequence; D002872:Chromosome Deletion; D003001:Cloning, Molecular / Q000379:methods; D004768:Enterotoxins / Q000235:genetics* / Q000302:isolation & purification / Q000633:toxicity; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D005798:Genes, Bacterial; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D011498:Protein Precursors / Q000235:genetics*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000633:toxicity
D001427:Bacterial Toxins; D004768:Enterotoxins; D029968:Escherichia coli Proteins; D011498:Protein Precursors; D011994:Recombinant Proteins; C034151:heat stable toxin (E coli)