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Cloning and expression of FomA, the major outer-membrane protein gene from Fusobacterium nucleatum T18
Abstract:
The major outer-membrane protein. FomA, of Fusobacterium nucleatum has been associated with porin activity, interbacterial adherence and stimulation of host immune cells. Until now, molecular analysis of FomA has not been possible because previous attempts to clone the fomA gene were not successful. The inability to clone F. nucleatum genes led to speculation that Escherichia coli may not be a suitable host. This report concerns the amplification of the fomA gene of F. nucleatum T18 using oligonucleotide primers containing restriction endonuclease sites that allow cloning of fomA into the E. coli expression vector pMMB67. The resultant plasmid, pXWI, was transformed into E. coli DH5 alpha, providing high-level expression of recombinant FomA (rFomA). Amino acid sequencing of rFomA demonstrated that the FomA signal peptide was correctly processed by E. coli signal peptidase I. rFomA was correctly localized to the outer membrane by the E. coli export pathway. The rFomA protein also displayed the heat-modifiable oligomeric and conformational properties of native FomA (nFomA). This demonstration of rFomA expression, processing, export, and secondary and tertiary structure in E. coli provides support for the feasibility of molecular analysis of the structure and function of FomA and other F. nucleatum proteins using recombinant techniques.
Insights
Researchers successfully cloned and expressed the major outer-membrane protein FomA from Fusobacterium nucleatum in E. coli. This breakthrough enables detailed molecular analysis of FomA structure and function, advancing our understanding of this important bacterial protein.
Area of Science:
- Microbiology
- Molecular Biology
- Protein Expression
Background:
- Fusobacterium nucleatum's major outer-membrane protein, FomA, is implicated in porin activity, bacterial adherence, and immune response.
- Previous attempts to clone the fomA gene were unsuccessful, hindering molecular analysis.
- This limitation led to speculation about the suitability of Escherichia coli as a host for F. nucleatum gene cloning.
Purpose of the Study:
- To amplify and clone the fomA gene from F. nucleatum T18 into an E. coli expression vector.
- To achieve high-level expression of recombinant FomA (rFomA) in E. coli.
- To confirm the correct processing, localization, and structural properties of rFomA in E. coli.
Main Methods:
- Amplification of the fomA gene using specific oligonucleotide primers with restriction sites.
- Cloning of the amplified fomA gene into the E. coli expression vector pMMB67, creating plasmid pXWI.
- Transformation of E. coli DH5 alpha with pXWI for high-level expression of rFomA.
- Amino acid sequencing and analysis of rFomA localization and conformational properties.
Main Results:
- Successful amplification and cloning of the fomA gene.
- High-level expression of recombinant FomA (rFomA) in E. coli.
- Demonstration of correct signal peptide processing by E. coli signal peptidase I.
- Correct outer membrane localization of rFomA via the E. coli export pathway.
- rFomA exhibited heat-modifiable oligomeric and conformational properties similar to native FomA (nFomA).
Conclusions:
- The successful expression, processing, and export of functional rFomA in E. coli validates E. coli as a suitable host for F. nucleatum gene cloning.
- This study provides a foundation for detailed molecular analysis of FomA structure and function using recombinant techniques.
- The findings pave the way for investigating other F. nucleatum proteins through similar recombinant approaches.