Gene fusions using the ompA gene coding for a major outer-membrane protein of Escherichia coli K12.
It has been shown previously that fragments of the Escherichia coli major outer membrane protein OmpA lacking CO2H-terminal parts can be incorporated into this membrane in vivo [Bremer et al. (1982) Eur. J. Biochem. 122, 223-231]. The possibility that these fragments can be used, via gene fusions, as vehicles to transport other proteins to the outer membrane has been investigated. To test whether fragments of a certain size were optimal for this purpose a set of plasmids was prepared encoding 160, 193, 228, 274, and 280 NH2-terminal amino acids of the 325-residue OmpA protein. The 160-residue fragment was not assembled into the outer membrane whereas the others were all incorporated with equal efficiencies. Thus, if any kind of OmpA-associated stop transfer is required during export the corresponding signal might be present between residues 160 and 193 but not CO2H-terminal to 193. The ompA gene was fused to the gene (tet) specifying tetracycline resistance and the gene for the major antigen (vp1) of foot-and-mouth disease virus. In the former case a 584-residue chimeric protein is encoded consisting NH2-terminally of 228 OmpA residues followed by 356 CO2H-terminal residues of the 396-residue 'tetracycline resistance protein'. In the other case the same part of OmpA is followed by 250 CO2H-terminal residues of the 213-residue Vp1 plus 107 residues partly derived from another viral protein and from the vector. Full expression of both hybrids proved to be lethal. Lipophilic sequences bordered by basic residues, present in the non-OmpA parts of both hybrids were considered as candidates for the lethal effect. A plasmid was constructed which codes for 280 OmpA residues followed by a 31-residue tail containing the sequence: -Phe-Val-Ile-Met-Val-Ile-Ala-Val-Ser-Cys-Lys-. Expression of this hybrid gene was lethal but by changing the reading frame for the tail to encode another, 30-residue sequence the deleterious effect was abolished. It is possible that the sequence incriminated acts as a stop signal for transfer through the plasma membrane thereby jamming export sites for other proteins and causing lethality. If so, OmpA appears to cross the plasma membrane completely during export.
D005537:Aphthovirus / Q000235:genetics; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D055598:Chemical Phenomena; D002621:Chemistry; D004352:Drug Resistance, Microbial; D004926:Escherichia coli / Q000235:genetics*; D005815:Genetic Code; D005821:Genetic Techniques; D005838:Genotype; D008565:Membrane Proteins / Q000235:genetics*; D010446:Peptide Fragments / Q000302:isolation & purification; D010455:Peptides / Q000302:isolation & purification; D010957:Plasmids; D021382:Protein Sorting Signals; D013754:Tetracyclines / Q000494:pharmacology
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D008565:Membrane Proteins; D010446:Peptide Fragments; D010455:Peptides; D021382:Protein Sorting Signals; D013754:Tetracyclines