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Molecular cloning and characterization of protective outer membrane protein P.69 from Bordetella pertussis
I G Charles1, G Dougan, D Pickard
1Wellcome Research Laboratories, Beckenham, Kent, England.
Abstract:
Protein P.69 is localized on the outer membrane of Bordetella pertussis and is one of the virulence factors believed to contribute to the disease state of whooping cough. We demonstrate that protein synthesis of P.69 is under genetic control of the vir locus. Using oligonucleotide probes derived from the protein sequence of a cyanogen bromide fragment, we have cloned the gene for P.69 from B. pertussis CN2992. Analysis of the DNA sequence reveals a G + C-rich gene capable of encoding a protein of 910 amino acids with a Mr of 93,478, suggesting that P.69 is a processed form of a larger precursor. In common with some of the genes in the pertussis toxin operon, the sequence CCTGG was found 5' to the ATG initiation codon. At the 3' end, 29 bases after the TAA stop codon, the sequence GTTTTTCCT was found and may have some function in transcription termination. A full-length clone of the gene for P.69 carried by the cosmid pBPI69 was unable to direct the expression of P.69 protein in an Escherichia coli host. The generation of P.69-fusion products allowed the detection of P.69-specific protein products synthesized in E. coli.
Insights
Researchers cloned the Bordetella pertussis P.69 gene, identifying its genetic control and sequence. While expression in E. coli was initially unsuccessful, fusion products enabled P.69 protein detection, advancing whooping cough research.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- Protein P.69 is an outer membrane protein of Bordetella pertussis.
- P.69 is a key virulence factor implicated in whooping cough pathogenesis.
- Its synthesis is regulated by the vir locus.
Purpose of the Study:
- To clone and characterize the gene encoding P.69 from Bordetella pertussis.
- To investigate the genetic regulation and sequence of P.69.
- To explore P.69 expression in a heterologous host system.
Main Methods:
- Oligonucleotide probes were used to clone the P.69 gene.
- DNA sequencing was performed to analyze the gene's structure.
- Gene expression studies in Escherichia coli, including the use of fusion proteins, were conducted.
Main Results:
- The P.69 gene was successfully cloned from B. pertussis CN2992.
- Sequence analysis revealed a G+C-rich gene encoding a 910-amino acid precursor protein.
- Regulatory sequences (CCTGG and GTTTTTCCT) were identified near the initiation and termination codons.
- Full-length P.69 expression in E. coli was not achieved, but P.69-specific products were detected using fusion proteins.
Conclusions:
- The P.69 gene and its regulatory elements have been identified.
- P.69 is likely a processed form of a larger precursor protein.
- Heterologous expression of P.69 in E. coli presents challenges, but fusion strategies allow for detection.