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Stable cloning of the amino terminus of the 60k outer membrane protein of Chlamydia trachomatis serovar L1

Stable cloning of the amino terminus of the 60k outer membrane protein of Chlamydia trachomatis serovar L1
Stable cloning of the amino terminus of the 60k outer membrane protein of Chlamydia trachomatis serovar L1
A mixed twenty-five base oligonucleotide probe was synthesized to the amino terminus of the 60 kDa cysteine-rich outer membrane protein of Chlamydia trachomatis serovar F. This probe hybridized to a single band in Southern blots of C. trachomatis serovar L1 DNA. However, repeated attempts at screening plasmid-based genomic libraries failed to yield positive signals. As an alternative approach, bacteriophage M13 was used as the primary cloning vector. With this vector two identical stable recombinants were isolated carrying a 2.2 kb Pst1 insert that hybridized to the oligonucleotide probe. Attempts to re-clone this fragment in plasmid-based vectors yielded very small unstable colonies. DNA encoding the amino terminus of the 60 kDa outer membrane protein was further localised to a 274 bp Sau3A fragment which was sub-cloned in both orientations in m13. DNA sequence analysis of this fragment from C. trachomatis serovar L1 demonstrated a perfect 15/15 amino acid match to the amino terminus of the 60 kDa outer membrane protein from C. trachomatis serovar F. On the basis of these results we suggest bacteriophage M13 as a general cloning vector to avoid the toxic effects of the amino termini of foreign outer membrane proteins in Escherichia coli.
chlamydia trachomatis, outer membrane protein, cloning, amino terminus
0378-1097
81-85
Clarke, I.N.
ff6c9324-3547-4039-bb2c-10c0b3327a8b
Lambden, P.R.
e99ecc21-50d7-4a43-9e79-efba46592c77
Clarke, I.N.
ff6c9324-3547-4039-bb2c-10c0b3327a8b
Lambden, P.R.
e99ecc21-50d7-4a43-9e79-efba46592c77

Clarke, I.N. and Lambden, P.R. (1988) Stable cloning of the amino terminus of the 60k outer membrane protein of Chlamydia trachomatis serovar L1. FEMS Microbiology Letters, 51 (1), 81-85. (doi:10.1111/j.1574-6968.1988.tb02973.x).

Record type: Article

Abstract

A mixed twenty-five base oligonucleotide probe was synthesized to the amino terminus of the 60 kDa cysteine-rich outer membrane protein of Chlamydia trachomatis serovar F. This probe hybridized to a single band in Southern blots of C. trachomatis serovar L1 DNA. However, repeated attempts at screening plasmid-based genomic libraries failed to yield positive signals. As an alternative approach, bacteriophage M13 was used as the primary cloning vector. With this vector two identical stable recombinants were isolated carrying a 2.2 kb Pst1 insert that hybridized to the oligonucleotide probe. Attempts to re-clone this fragment in plasmid-based vectors yielded very small unstable colonies. DNA encoding the amino terminus of the 60 kDa outer membrane protein was further localised to a 274 bp Sau3A fragment which was sub-cloned in both orientations in m13. DNA sequence analysis of this fragment from C. trachomatis serovar L1 demonstrated a perfect 15/15 amino acid match to the amino terminus of the 60 kDa outer membrane protein from C. trachomatis serovar F. On the basis of these results we suggest bacteriophage M13 as a general cloning vector to avoid the toxic effects of the amino termini of foreign outer membrane proteins in Escherichia coli.

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Published date: 1 June 1988
Keywords: chlamydia trachomatis, outer membrane protein, cloning, amino terminus
Organisations: Faculty of Medicine

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Local EPrints ID: 353702
URI: https://eprints.soton.ac.uk/id/eprint/353702
ISSN: 0378-1097
PURE UUID: e61036c4-05c2-474c-947a-a688e8016b95
ORCID for I.N. Clarke: ORCID iD orcid.org/0000-0002-4938-1620

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Date deposited: 14 Jun 2013 10:25
Last modified: 06 Jun 2018 13:19

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Author: I.N. Clarke ORCID iD
Author: P.R. Lambden

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