A new membrane-bound OprI lipoprotein expression vector. High production of heterologous fusion proteins in gram (-) bacteria and the implications for oral vaccination.
We have previously described the development of cloning vectors for the production of OprI-based outer membrane fusion proteins in E. coli (Cornelis et al., 1996) and now describe the construction of a new vector, containing a lacI(q) gene, resulting in tight repression of the promotor and allowing its use in other Gram (-) bacteria. The new pVUB3 expression vector encodes a truncated but active LacI(q)(341) repressor which binds to the single operator in the vector. A high repression of the trc promotor was observed, resulting in a very low basal leakage of expression and very high production levels of OprI or derivatives after IPTG induction in E. coli. Bacterial viability was not affected under uninduced conditions, but the number of viable cell counts decreased after production of large amounts of the outer membrane-bound OprI lipoprotein and its derivatives, both in E. coli and Salmonella typhimurium. This highly repressible system allows us to extend the use of OprI vectors in other Gram (-) bacteria, resulting in the production of outer membrane-bound lipid-modified molecules, opening the possibility for its application in the design of potential live Salmonella-based subunit vaccines.
Cote-Sierra
J
J
Department of Immunology, Parasitology and Ultrastructure, Flanders Interuniversity Institute for Biotechnology, Vrije Universiteit Brussel, Sint Genesius Rode, Belgium.
Jongert
E
E
Bredan
A
A
Gautam
D C
DC
Parkhouse
M
M
Cornelis
P
P
De Baetselier
P
P
Revets
H
H
D000595:Amino Acid Sequence; D000818:Animals; D000953:Antigens, Protozoan / Q000235:genetics; D000954:Antigens, Surface / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics*; D001483:Base Sequence; D001692:Biological Transport; D002213:Capsid / Q000235:genetics / Q000276:immunology; D036022:Capsid Proteins; D002455:Cell Division / Q000187:drug effects; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000166:cytology / Q000187:drug effects / Q000235:genetics; D029968:Escherichia coli Proteins; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects; D005822:Genetic Vectors / Q000187:drug effects / Q000235:genetics* / Q000276:immunology; D006090:Gram-Negative Bacteria / Q000235:genetics*; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D056968:Lac Repressors; D018320:Leishmania major / Q000235:genetics; D008074:Lipoproteins / Q000235:genetics*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000235:genetics / Q000276:immunology; D010957:Plasmids / Q000187:drug effects / Q000235:genetics; D011401:Promoter Regions, Genetic; D015800:Protozoan Proteins; D011993:Recombinant Fusion Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics; D012486:Salmonella typhimurium / Q000235:genetics / Q000276:immunology; D014614:Vaccines, Synthetic
D000953:Antigens, Protozoan; D000954:Antigens, Surface; D001426:Bacterial Proteins; D036022:Capsid Proteins; D029968:Escherichia coli Proteins; C094837:FMDV 15 peptide; D056968:Lac Repressors; C539064:LacI protein, E coli; D008074:Lipoproteins; D010446:Peptide Fragments; D015800:Protozoan Proteins; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D014614:Vaccines, Synthetic; C060507:outer membrane lipoprotein I, Bacteria; C072094:surface antigen P2, Leishmania; D007544:Isopropyl Thiogalactoside