Fimbriae of Bacteroides nodosus: protein engineering of the structural subunit for the production of an exogenous peptide.
The pattern of sequence variation between Bacteroides nodosus fimbrial subunits of different serotypes suggests a degree of flexibility, which might be exploited for protein engineering approaches for the expression of other peptides. We have tested this using the well-characterized peptide epitope from VP1 of foot-and-mouth disease virus (FMDV), residues 144-159: LRGDLQVLAQKVARTL (strain 01-BFS). Using bacterial codon usage, several oligonucleotides were designed for the substitution of this sequence internally at hypervariable regions of the fimbrial subunit (aligned for maximum homology), and for its addition at the carboxyterminus with a diglycine spacer as a flexible hinge. Following site-directed mutagenesis in phage M13, the modified genes were placed under PL promoter control and placed in a broad host range vector. Analysis of the variant proteins expressed in E. coli showed that these substitutions affected, to varying extents, recognition by both anti-fimbrial and anti-FMDV antibodies, indicating that hypervariable region 2 is a major antigenic determinant of the fimbrial subunit and that local stereochemical effects can influence antibody binding to the FMDV peptide antigenic determinant. In Pseudomonas aeruginosa, viable transformants could only be obtained with the mutant gene encoding the carboxy-terminal graft. These cells provided fimbrial preparations comprised of the modified subunit. This then constitutes the prototype for the development of a general expression system for the production of vaccine epitopes and other bioactive peptides. Furthermore, there is considerable scope for further modification of the system, for example by engineering specific chemical or protease cleavage sites for release of the grafted peptide. Alternatively, the fimbriae themselves may serve as a useful supramolecular carrier or adjuvant for immune provocation.
Jennings
P A
PA
CSIRO Division of Biotechnology, Laboratory for Molecular Biology, North Ryde, NSW, Australia.
Bills
M M
MM
Irving
D O
DO
Mattick
J S
JS
D000595:Amino Acid Sequence; D001439:Bacteroides; D015153:Blotting, Western; D004251:DNA Transposable Elements; D004591:Electrophoresis, Polyacrylamide Gel; D010861:Fimbriae, Bacterial; D008969:Molecular Sequence Data; D010455:Peptides; D015202:Protein Engineering
D004251:DNA Transposable Elements; D010455:Peptides