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Stable expression of meningococcal class 1 protein in an antigenically reactive form in outer membranes of Escherichia coli

Stable expression of meningococcal class 1 protein in an antigenically reactive form in outer membranes of Escherichia coli
Stable expression of meningococcal class 1 protein in an antigenically reactive form in outer membranes of Escherichia coli
The entire gene encoding the class 1 outer membrane protein of Neisseria meningitidis is located on a 2.2 kb fragment, obtained on digestion of chromosomal DNA with Xbal. This Xbal fragment from strain MC50 (subtype P1-16), which had previously been cloned in bacteriophage M13, has been transferred to the plasmid vector pMTL20. The resulting plasmid (pPORA100) was propagated in Escherichia coli (JM109) and cell lysates were subjected to SDS-PAGE. Western blotting with anti-class 1 protein antibodies revealed constitutive expression of a protein of 41kD, corresponding to the class 1 protein of the parent meningococcal strain, which was absent in the E. coli control Fractionation of E. coli cells carrying the recombinant plasmid revealed that the protein was exclusively located in the outer membrane, and N-terminal amino acid analysis of the expressed protein revealed that normal processing of the signal peptide had occurred. Immuno-gold electron microscopy showed that the protective epitope recognized by a P1-16 subtype-specific monoclonal antibody was exposed in an antigenically reactive form on the surface of E. coli cells carrying plasmid pPORA100. In contrast, expression in E. coli of a second plasmid (pPORA104) lacking the coding sequence for the first 15 amino acids of the signal peptide resulted in accumulation of recombinant class 1 protein only in the cytoplasm of the cells.

Thus the presence of the meningococcal signal sequence ensures expression of this meningococcal porin protein in an antigenically native conformation in outer membranes of E. coli, while its absence results in expression of a soluble protein. Such constructs illustrate the potential use of recombinant DNA technology for the development of effective human vaccines against meningococcal infection.
0950-382X
769-776
White, D.A.
4a561727-96db-407d-ae0a-a18438707c6c
Barlow, A.K.
37084842-5493-4cf1-8405-1d452aa51336
Clarke, I.N.
ff6c9324-3547-4039-bb2c-10c0b3327a8b
Heckels, J.E.
fcfcfafe-5ca8-4728-9c5e-cb67f9af7e31
White, D.A.
4a561727-96db-407d-ae0a-a18438707c6c
Barlow, A.K.
37084842-5493-4cf1-8405-1d452aa51336
Clarke, I.N.
ff6c9324-3547-4039-bb2c-10c0b3327a8b
Heckels, J.E.
fcfcfafe-5ca8-4728-9c5e-cb67f9af7e31

White, D.A., Barlow, A.K., Clarke, I.N. and Heckels, J.E. (1990) Stable expression of meningococcal class 1 protein in an antigenically reactive form in outer membranes of Escherichia coli. Molecular Microbiology, 4 (5), 769-776. (PMID:2117694)

Record type: Article

Abstract

The entire gene encoding the class 1 outer membrane protein of Neisseria meningitidis is located on a 2.2 kb fragment, obtained on digestion of chromosomal DNA with Xbal. This Xbal fragment from strain MC50 (subtype P1-16), which had previously been cloned in bacteriophage M13, has been transferred to the plasmid vector pMTL20. The resulting plasmid (pPORA100) was propagated in Escherichia coli (JM109) and cell lysates were subjected to SDS-PAGE. Western blotting with anti-class 1 protein antibodies revealed constitutive expression of a protein of 41kD, corresponding to the class 1 protein of the parent meningococcal strain, which was absent in the E. coli control Fractionation of E. coli cells carrying the recombinant plasmid revealed that the protein was exclusively located in the outer membrane, and N-terminal amino acid analysis of the expressed protein revealed that normal processing of the signal peptide had occurred. Immuno-gold electron microscopy showed that the protective epitope recognized by a P1-16 subtype-specific monoclonal antibody was exposed in an antigenically reactive form on the surface of E. coli cells carrying plasmid pPORA100. In contrast, expression in E. coli of a second plasmid (pPORA104) lacking the coding sequence for the first 15 amino acids of the signal peptide resulted in accumulation of recombinant class 1 protein only in the cytoplasm of the cells.

Thus the presence of the meningococcal signal sequence ensures expression of this meningococcal porin protein in an antigenically native conformation in outer membranes of E. coli, while its absence results in expression of a soluble protein. Such constructs illustrate the potential use of recombinant DNA technology for the development of effective human vaccines against meningococcal infection.

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Published date: May 1990
Organisations: Faculty of Medicine

Identifiers

Local EPrints ID: 352635
URI: https://eprints.soton.ac.uk/id/eprint/352635
ISSN: 0950-382X
PURE UUID: 1fc74903-105e-4318-9989-63491bc331fe
ORCID for I.N. Clarke: ORCID iD orcid.org/0000-0002-4938-1620

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Date deposited: 04 Jun 2013 11:06
Last modified: 06 Jun 2018 13:19

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Contributors

Author: D.A. White
Author: A.K. Barlow
Author: I.N. Clarke ORCID iD
Author: J.E. Heckels

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