Cloning, sequencing and expression of an Eikenella corrodens gene encoding a component protein of the lectin-like adhesin complex.
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), 35748). 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.
Yumoto
H
H
Department of Conservative Dentistry, Tokushima University School of Dentistry, Japan. yumoto@dent.dent.tokushima-u.ac.jp
Azakami
H
H
Nakae
H
H
Matsuo
T
T
Ebisu
S
S
D000595:Amino Acid Sequence; D001422:Bacterial Adhesion / Q000235:genetics*; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D001483:Base Sequence; D003001:Cloning, Molecular; D016124:Eikenella corrodens / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial / Q000235:genetics*; D037102:Lectins; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D015183:Restriction Mapping; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
D001425:Bacterial Outer Membrane Proteins; D037102:Lectins; C103685:PorA protein, Eikenella; D011993:Recombinant Fusion Proteins