Cloning and expression of FomA, the major outer-membrane protein gene from Fusobacterium nucleatum T18.
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.
Haake
S K
SK
Section of Periodontics, UCLA School of Dentistry 90095-1668, USA.
Wang
X
X
D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D016967:Fusobacterium nucleatum / Q000235:genetics*; D015870:Gene Expression; D005798:Genes, Bacterial; D005822:Genetic Vectors; D006358:Hot Temperature; D008969:Molecular Sequence Data; D008970:Molecular Weight; D018272:Porins; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis
D001425:Bacterial Outer Membrane Proteins; D018272:Porins; D011993:Recombinant Fusion Proteins; C083760:fomA protein, Fusobacterium