Hybrid enterotoxin LTA::STa proteins and their protection from degradation by in vivo association with B-subunits of Escherichia coli heat-labile enterotoxin.
Chimeric proteins exhibiting antigenic determinants of the heat-labile enterotoxin (LT) and heat-stable (STa) enterotoxins on the same molecule may provide a means to obtain immunoprophylactic and diagnostic reagents for Escherichia coli-caused diarrhea. We recently showed that fusion of two different lengths of the STa gene to the C end of the A-subunit of LT (LTA) results in LTA::STa fusion proteins as monitored by GM1-ELISA [Sanchez et al.: FEBS Lett. 208 (1986) 194-198]. Here we determine the approximate molecular size of the LTA::STa fusion proteins and provide further evidence of their hybrid nature by immunoblot analysis. Using this technique we also demonstrate that to obtain detectable amounts of these recombinant proteins it is essential to coexpress them with the respective B-subunit of LT (LTB). We propose that this dependence on coexpression reflects the association between the LTA::STa hybrids and LTB subunits. The resulting LTA::STa/LTB complexes were found in the E. coli periplasm. This indicated that the exported hybrids, once associated with LTB, were stabilized and formed molecules that behaved essentially as native LT. The protective effect exerted by the B-subunit might conceivably be extended to other LTA-derived hybrid proteins, thus allowing the fusion of other foreign peptides to LTA and their subsequent recovery in the same fashion.
Sanchez
J
J
Department of Medical Microbiology, University of Göteborg, Sweden.
Hirst
T R
TR
Uhlin
B E
BE
D000595:Amino Acid Sequence; D001427:Bacterial Toxins / Q000235:genetics*; D001483:Base Sequence; D004262:DNA Restriction Enzymes; D004768:Enterotoxins / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D005796:Genes; D005798:Genes, Bacterial; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D010957:Plasmids; D055503:Protein Multimerization
D001427:Bacterial Toxins; D004768:Enterotoxins; D029968:Escherichia coli Proteins; D046911:Macromolecular Substances; C034151:heat stable toxin (E coli); C027538:heat-labile enterotoxin, E coli; D004262:DNA Restriction Enzymes