Stable cloning of the amino terminus of the 60 kDa outer membrane protein ofChlamydia trachomatisserovar L1

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.