PERMISSIBLE PEPTIDE INSERTIONS SURROUNDING THE SIGNAL PEPTIDE MATURE PROTEIN JUNCTION OF THE CLPG PREPILIN - CS31A FIMBRIAE OF ESCHERICHIA-COLI AS CARRIERS OF FOREIGN SEQUENCES

The clpG gene, expressing the Escherichia coli major CS31A fimbrial su bunit ClpG, was subjected to random mutagenesis by insertion of an Eco RI linker and a kanamycin-resistance (Km(R)) cassette into the multipl e newly generated EcoRI sites. The Km(R) gene was then excised by PstI , which left a 48-bp linker representing the heterologous sequence. Th e same procedure was followed to introduce a synthetic oligodeoxyribon ucleotide (oligo) corresponding to epitope C from the spike protein S from the porcine transmissible gastroenteritis coronavirus (TGEV). Nin e insertion/deletion mutants (indels) that contained long foreign pept ides variously located around the ClpG signal peptide (SP) processing site were characterized. A striking feature of this study is the varie ty of amino acid (aa) insertions in the ClpG prepilin that have little or no effect on CS31A fimbria biogenesis. These 'permissive' sites to lerate inserts of 18 or 19 aa and accept sequences of different nature s in view of their aa composition, charge and hydrophobicity. The resu lts obtained here are also interesting in light of the high level of a a sequence conservation seen in the SP and N-terminal domains of the C lpG-related subunits. The structure-function relationship of the ClpG SP is discussed. The TGEV-C epitope fused to the N-terminal end of the mature ClpG protein was cell-surface exposed, as observed on immune-e lectron microscopy. Therefore, the CS31A fimbria seems to be a potent tool for the presentation of foreign antigenic determinants or the pro duction of heterologous polypeptides in E. coli.