The Borrelia burgdorferi flagellum-associated 41-kilodalton antigen (flagellin): molecular cloning, expression, and amplification of the gene.
Monoclonal antibodies directed against the major Borrelia burgdorferi flagellar protein, the 41-kilodalton (kDa) protein flagellin, were used to monitor cloning and expression of the flagellin gene from a Borrelia burgdorferi genomic library. The structure of the gene was analyzed, and recombinant nonfusion flagellin was produced in Escherichia coli. A DNA sequence analysis of the 41-kDa flagellin gene revealed the presence of an open reading frame that encoded a protein having 336 amino acid residues and a calculated molecular mass of 35.8 kDa, indicating that there was posttranslational modification of the natural 41-kDa flagellin protein. Upstream from the AUG start codon sequence we identified motifs corresponding to consensus procaryotic promoter elements which could be utilized by the cloned flagellin gene when it was expressed in E. coli MC1061. The deduced flagellin protein sequence exhibited high levels of homology to sequences of flagellin proteins from Bacillus subtilis and Salmonella typhimurium. The levels of sequence similarity for the amino- and carboxy-terminal portions were about 65 and 56%, respectively. DNA sequence information on the flagellin gene was used to design oligonucleotides for gene amplification by the polymerase chain reaction method, and by using this method 0.01 pg of Borrelia burgdorferi DNA could be detected. Our results provide a basis for further biochemical analysis of the 41-kDa flagellin protein, investigation of the role of this protein in host-pathogen interactions, and development of a standardized reagent for diagnostic systems for Borrelia burgdorferi infections.
Wallich
R
R
Department of Applied Immunology, German Cancer Research Center, Heidelberg, Federal Republic of Germany.
Moter
S E
SE
Simon
M M
MM
Ebnet
K
K
Heiberger
A
A
Kramer
M D
MD
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology; D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D015748:Borrelia burgdorferi Group / Q000235:genetics* / Q000276:immunology; D003001:Cloning, Molecular; D003062:Codon; D005408:Flagellin / Q000235:genetics* / Q000276:immunology; D015870:Gene Expression; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000276:immunology; D015183:Restriction Mapping; D012995:Solubility
D000911:Antibodies, Monoclonal; D001426:Bacterial Proteins; D003062:Codon; D011994:Recombinant Proteins; D005408:Flagellin