key: cord-0943365-i3mebqtq authors: Der Vartanian, Maurice; Méchin, Marie-Claire; Jaffeux, Bernard; Bertin, Yolande; Félix, Isabelle; Gaillard-Martinie, Brigitte title: Permissible peptide insertions surrounding the signal peptide-mature protein junction of the ClpG prepilin: CS31A fimbriae of Escherichia coli as carriers of foreign sequences date: 1994-10-11 journal: Gene DOI: 10.1016/0378-1119(94)90229-1 sha: 95349ecdf1b234c3a53dfd1c8dc9364a340aa0fe doc_id: 943365 cord_uid: i3mebqtq Abstract The clpG gene, expressing the Escherichia coli major CS31A fimbrial subunit ClpG, was subjected to random mutagenesis by insertion of an EcoRI linker and a kanamycin-resistance (KmR) cassette into the multiple newly generated EcoRI sites. The KmR gene was then excised by PstI, which left a 48-bp linker representing the heterologous sequence. The same procedure was followed to introduce a synthetic oligodeoxyribonucleotide (oligo) corresponding to epitope C from the spike protein S from the porcine transmissible gastroenteritis coronavirus (TGEV). Nine insertion/deletion mutants (indels) that contained long foreign peptides variously located around the ClpG signal peptide (SP) processing site were characterized. A striking feature of this study is the variety of amino acid (aa) insertions in the ClpG prepilin that have little or no effect on CS31A fimbria biogenesis. These ‘permissive’ sites tolerate inserts of 18 or 19 aa and accept sequences of different natures in view of their aa composition, charge and hydrophobicity. The results obtained here are also interesting in light of the high level of aa sequence conservation seen in the SP and N-terminal domains of the ClpG-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 immuno-electron microscopy. Therefore, the CS31A fimbria seems to be a potent tool for the presentation of foreign antigenic determinants or the production of heterologous polypeptides in E. coli. the heterologous sequence. The same procedure was followed to introduce a synthetic oligodeoxyribonucleotide (oligo) corresponding to epitope C from the spike protein S from the porcine transmissible gastroenteritis coronavirus (TGEV). Nine insertion/deletion mutants (indels) that contained long foreign peptides variously located around the ClpG signal peptide (SP) processing site were characterized. A striking feature of this study is the variety of amino acid (aa) insertions in the ClpG prepilin that have little or no effect on CS31A fimbria biogenesis. These 'permissive' sites tolerate inserts of 18 or 19 aa and accept sequences of different natures in view of their aa composition, charge and hydrophobicity. The results obtained here are also interesting in light of the high level of aa sequence conservation seen in the SP and N-terminal domains of the ClpG-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 immuno-electron microscopy. Therefore, the CS31A fimbria seems to be a potent tool for the presentation of foreign antigenic determinants or the production of heterologous polypeptides in E. coli. INTRODUCTION Polymer CS31A is a plasmid-encoded fibrillar protein associated with animal and human pathogenic isolates of Escherichia coli (Contrepois et al., 1989; Cherifi et al., CS3 I A biogcncsis ( Martin ct al., I99 I ). They include the ma_jor ClpG monomer and several accessory proteins involvcd in the stabilization, transport and assembly of CIpG. Extensive nt sequence homology throughout the accessory protein-encoding genes of CS31 A. KHH and F41 operons has been demonstrated by Southern hybridization analysis (Casey et al., 1990 : Martin ct al.. 1991 Korth ct al., 1991 ) . The accessory systems mediating cxpression of these operons may be functionally interchangeable, indicating that CS3lA. K88 and F41 fimbriae arc members of a closely related family ( Korth et al.. 1992) . The c