Summary
Fusion proteins comprising the amino-terminal 99 amino acids of the bacteriophage MS2 replicase and various portions of OmpV a major outer membrane protein of Vibrio cholerae were expressed in Escherichia coli K12. These fusions were expressed under the control of the PL promoter of bacteriophage λ, and expression was controlled using a cIts repressor. Fusions occurring within the secretory signal sequence of OmpV gave rise to the production of mature OmpV. The efficiency, however, decreased with progressive deletion of the signal sequence within the fusions. The reactivity of various OmpV fusions with antisera raised against purified OmpV and whole bacteria demonstrated the existence of two antigenic domains: one present in the denatured form and another in the membrane-associated form of OmpV. These domains correspond to markedly hydrophilic regions of the protein as would be predicted for surface-exposed epitopes.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Achtman M, Schwuchow S, Helmuth R, Morelli G, Manning PA (1978) Cell-cell interactions in conjugating Escherichia coli: Con− mutants and stabilization of mating aggregates. Mol Gen Genet 164:171–183
Chen EY, Seeburg PH (1985) Supercoil sequencing: a fast and simple method for sequencing plasmid DNA. DNA 4:165–170
Hanahan D (1983) Studies on transformation of Escherichia coli with plasmids. J Mol Biol 166:557–580
Inouye S, Soberon X, Fanceschini T, Nakamura K, Hakura K, Inouye M (1982) Role of positive charge in the amino terminal region of the signal peptide in protein secretion across the membrane. Proc Natl Acad Sci USA 79:3438–3441
Klinkert MQ, Herrmann R, Schaller HE (1985) Surface proteins of Mycoplasma hyopneumoniae identified from an Escherichia coli expression plasmid library. Infect Immun 49:329–335
Kyte J, Doolittle RF (1982) A simple method for displaying the hydropathic character of a protein. J Mol Biol 157:105–132
Lugtenberg B, Meijers J, Peters R, Van der Hoek P, Van Alphen L (1975) Electrophoretic resolution of the ‘major outer membrane protein’ of Escherichia coli K-12 into four bands. FEBS Lett 58:254–258
Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning: A laboratory manual. Cold Spring Harbor Laboratory, New York
Manning PA, Imbesi F, Haynes DR (1982) Cell envelope proteins in Vibrio cholerae. FEMS Microbiol Lett 14:159–166
Manning PA, Haynes DR (1984) A common-immunogenic Vibrio outer membrane protein. FEMS Microbiol Lett 24:297–302
Manning PA, Stevenson G, Heuzenroeder MW (1986) Molecular cloning of a common major outer membrane protein of Vibrio cholerae. In: Takeda Y, Pierce N (eds) Advances in cholera and related diarrheas, vol 4. KTK Publications, Japan (in press)
Pohlner J, Meyer TF, Jalajakumari MB, Manning PA (1986) Nucleotide sequence of ompV, the gene for a major Vibrio cholerae outer membrane protein. Mol Gen Genet 205:494–500
Remaut E, Stanssens P, Fiers W (1981) Plasmid vectors for high efficiency expression controlled by the PL promoter of coliphage lambda. Gene 15:81–93
Remaut E, Tsae H, Fiers W (1983) Improved plasmid vectors with a thermoinducible expression and temperature regulated runaway replication. Gene 22:103–113
Sanger F, Coulson AR, Barrell BG, Smith AJH, Roe BA (1980) Cloning in single-stranded bacteriophage as an aid to rapid DNA sequencing. J Mol Biol 143:161–178
Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467
Stevenson G, Leavesley DI, Lagnado CA, Heuzenroeder MW, Manning PA (1985) Purification of the 25 kDal Vibrio cholerae major outer membrane protein and the molecular cloning of its gene: ompV. Eur J Biochem 148:385–390
Stevenson G, Manning PA (1985) Galactose epimeraseless (GalE) mutant G30 of Salmonella typhimurium is a good potential live oral vaccine carrier for fimbrial antigens. FEMS Microbiol Lett 28:317–321
Strebel K, Beck E, Strohmaier K, Schaller H (1986) Characterization of foot-and-mouth disease virus gene products with antisera against bacterially synthesized fusion proteins. J Virol 57:983–991
Towbin H, Staehlin T, Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76:4350–4354
Watson MEE (1984) Compilation of published signal sequences. Nucleic Acids Res 12:5145–5164
Author information
Authors and Affiliations
Additional information
Communicated by E.K.F. Bautz
Rights and permissions
About this article
Cite this article
Pohlner, J., Meyer, T.F. & Manning, P.A. Serological properties and processing in Escherichia coli K12 of OmpV fusion proteins of Vibrio cholerae . Mol Gen Genet 205, 501–506 (1986). https://doi.org/10.1007/BF00338089
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF00338089