Cloning, sequencing and expression of an Eikenella corrodens gene encoding a component protein of the lectin-like adhesin complex

Abstract

A lectin-like substance (LS), that was isolated from Eikenella corrodens (Ec) 1073, migrated as proteins of about 300 and 45 kDa upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. In this study, we cloned the gene encoding the 45-kDa protein and predicted its structure and function. Based on the N-terminal 23-amino acid (aa) sequence of this protein, we cloned the region for its N-terminus. We cloned the entire gene by means of gene walking using polymerase chain reaction and Southern hybridization. The nucleotide sequences of cloned fragments revealed an open reading frame encoding a polypeptide of 330 aa ( M r 35 748). This ORF displayed high homology to those of porins of Neisseria species. Using the T7-expression system, the 45-kDa protein was produced in E. coli. Our results suggested that the 45-kDa protein of Ec 1073 is a component of the EcLS complex, and that it is the major outer membrane protein.