Skip to main content
PMC home page
Applied and Environmental Microbiology logoLink to Applied and Environmental Microbiology
. 1989 Apr;55(4):984–993. doi: 10.1128/aem.55.4.984-993.1989

Cloning of DNA sequences encoding foreign peptides and their expression in the K88 pili.

G Thiry 1, A Clippe 1, T Scarcez 1, J Petre 1
PMCID: PMC184235  PMID: 2471451

Abstract

A genetic system that allows the cloning of a peptide-coding sequence in the Escherichia coli K88ac and K88ad pilin genes and their expression as recombinant pili has been constructed. Two insertion vectors were created by subcloning the pilin genes in a pBR322 plasmid and replacing the coding sequence of two nonconserved clusters by a linker. The K88ac helper genes were subcloned in the compatible pACYC184 plasmid, and expression of pili by bacteria carrying both plasmids occurred by complementation. Two peptide-coding sequences of the influenza hemagglutinin were cloned in both insertion vectors, and recombinant pilins were shown to be assembled in pili. One recombinant pilus was shown to elicit antibodies against the synthetic peptide in immunized rats. The somatostatin-coding sequence was cloned in both vectors and led in one case to detectable pilus production. The fused somatostatin was shown to be recognized by specific monoclonal and polyclonal antibodies.

Full text

PDF
984

Images in this article

988Image
on p.988
990Image
on p.990
990Image
on p.990

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bolivar F., Rodriguez R. L., Greene P. J., Betlach M. C., Heyneker H. L., Boyer H. W., Crosa J. H., Falkow S. Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene. 1977;2(2):95–113. [PubMed] [Google Scholar]
  2. CAMPBELL A. Sensitive mutants of bacteriophage lambda. Virology. 1961 May;14:22–32. doi: 10.1016/0042-6822(61)90128-3. [DOI] [PubMed] [Google Scholar]
  3. Chang A. C., Cohen S. N. Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J Bacteriol. 1978 Jun;134(3):1141–1156. doi: 10.1128/jb.134.3.1141-1156.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Dykes C. W., Halliday I. J., Read M. J., Hobden A. N., Harford S. Nucleotide sequences of four variants of the K88 gene of porcine enterotoxigenic Escherichia coli. Infect Immun. 1985 Oct;50(1):279–283. doi: 10.1128/iai.50.1.279-283.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Foged N. T., Klemm P., Elling F., Jorsal S. E., Zeuthen J. Monoclonal antibodies to K88ab, K88ac and K88ad fimbriae from enterotoxigenic Escherichia coli. Microb Pathog. 1986 Feb;1(1):57–69. doi: 10.1016/0882-4010(86)90032-x. [DOI] [PubMed] [Google Scholar]
  6. Green N., Alexander H., Olson A., Alexander S., Shinnick T. M., Sutcliffe J. G., Lerner R. A. Immunogenic structure of the influenza virus hemagglutinin. Cell. 1982 Mar;28(3):477–487. doi: 10.1016/0092-8674(82)90202-1. [DOI] [PubMed] [Google Scholar]
  7. Grosjean H., Fiers W. Preferential codon usage in prokaryotic genes: the optimal codon-anticodon interaction energy and the selective codon usage in efficiently expressed genes. Gene. 1982 Jun;18(3):199–209. doi: 10.1016/0378-1119(82)90157-3. [DOI] [PubMed] [Google Scholar]
  8. Gyles C. L., Maas W. K. Recombinant DNA technology and enterotoxigenic Escherichia coli vaccines. Prog Vet Microbiol Immunol. 1987;3:139–158. [PubMed] [Google Scholar]
  9. Itakura K., Hirose T., Crea R., Riggs A. D., Heyneker H. L., Bolivar F., Boyer H. W. Expression in Escherichia coli of a chemically synthesized gene for the hormone somatostatin. Science. 1977 Dec 9;198(4321):1056–1063. doi: 10.1126/science.412251. [DOI] [PubMed] [Google Scholar]
  10. Jacobs A. A., Venema J., Leeven R., van Pelt-Heerschap H., de Graaf F. K. Inhibition of adhesive activity of K88 fibrillae by peptides derived from the K88 adhesin. J Bacteriol. 1987 Feb;169(2):735–741. doi: 10.1128/jb.169.2.735-741.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Jou W. M., Verhoeyen M., Devos R., Saman E., Fang R., Huylebroeck D., Fiers W., Threlfall G., Barber C., Carey N. Complete structure of the hemagglutinin gene from the human influenza A/Victoria/3/75 (H3N2) strain as determined from cloned DNA. Cell. 1980 Mar;19(3):683–696. doi: 10.1016/s0092-8674(80)80045-6. [DOI] [PubMed] [Google Scholar]
  12. Kehoe M., Winther M., Dougan G. Expression of a cloned K88ac adhesion antigen determinant: identification of a new adhesion cistron and role of a vector-encoded promoter. J Bacteriol. 1983 Sep;155(3):1071–1077. doi: 10.1128/jb.155.3.1071-1077.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Klemm P. Fimbrial adhesions of Escherichia coli. Rev Infect Dis. 1985 May-Jun;7(3):321–340. doi: 10.1093/clinids/7.3.321. [DOI] [PubMed] [Google Scholar]
  14. Krogfelt K. A., Meldal M., Klemm P. K88 fimbrial antigens: identification of antigenic determinants by the use of synthetic peptides. Microb Pathog. 1987 Jun;2(6):465–472. doi: 10.1016/0882-4010(87)90053-2. [DOI] [PubMed] [Google Scholar]
  15. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  16. Maxam A. M., Gilbert W. A new method for sequencing DNA. Proc Natl Acad Sci U S A. 1977 Feb;74(2):560–564. doi: 10.1073/pnas.74.2.560. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Meselson M., Yuan R. DNA restriction enzyme from E. coli. Nature. 1968 Mar 23;217(5134):1110–1114. doi: 10.1038/2171110a0. [DOI] [PubMed] [Google Scholar]
  18. Messing J. New M13 vectors for cloning. Methods Enzymol. 1983;101:20–78. doi: 10.1016/0076-6879(83)01005-8. [DOI] [PubMed] [Google Scholar]
  19. Mooi F. R., Claassen I., Bakker D., Kuipers H., de Graaf F. K. Regulation and structure of an Escherichia coli gene coding for an outer membrane protein involved in export of K88ab fimbrial subunits. Nucleic Acids Res. 1986 Mar 25;14(6):2443–2457. doi: 10.1093/nar/14.6.2443. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Mooi F. R., de Graaf F. K. Molecular biology of fimbriae of enterotoxigenic Escherichia coli. Curr Top Microbiol Immunol. 1985;118:119–138. doi: 10.1007/978-3-642-70586-1_7. [DOI] [PubMed] [Google Scholar]
  21. Rüther U., Koenen M., Sippel A. E., Müller-Hill B. Exon cloning: immunoenzymatic identification of exons of the chicken lysozyme gene. Proc Natl Acad Sci U S A. 1982 Nov;79(22):6852–6855. doi: 10.1073/pnas.79.22.6852. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Stahl H. D., Coppel R. L., Brown G. V., Saint R., Lingelbach K., Cowman A. F., Anders R. F., Kemp D. J. Differential antibody screening of cloned Plasmodium falciparum sequences expressed in Escherichia coli: procedure for isolation of defined antigens and analysis of human antisera. Proc Natl Acad Sci U S A. 1984 Apr;81(8):2456–2460. doi: 10.1073/pnas.81.8.2456. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Stirm S., Orskov F., Orskov I., Mansa B. Episome-carried surface antigen K88 of Escherichia coli. II. Isolation and chemical analysis. J Bacteriol. 1967 Feb;93(2):731–739. doi: 10.1128/jb.93.2.731-739.1967. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Stüber D., Bujard H. Organization of transcriptional signals in plasmids pBR322 and pACYC184. Proc Natl Acad Sci U S A. 1981 Jan;78(1):167–171. doi: 10.1073/pnas.78.1.167. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Vincent S. R., McIntosh C. H., Buchan A. M., Brown J. C. Central somatostatin systems revealed with monoclonal antibodies. J Comp Neurol. 1985 Aug 8;238(2):169–186. doi: 10.1002/cne.902380205. [DOI] [PubMed] [Google Scholar]

Articles from Applied and Environmental Microbiology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES