PMID:7523252
| Citation |
Der Vartanian, M, Méchin, MC, Jaffeux, B, Bertin, Y, Félix, I and Gaillard-Martinie, B (1994) Permissible peptide insertions surrounding the signal peptide-mature protein junction of the ClpG prepilin: CS31A fimbriae of Escherichia coli as carriers of foreign sequences. Gene 148:23-32 |
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| 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. |
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| Keywords |
Amino Acid Sequence; Animals; Antigens, Bacterial; Bacterial Outer Membrane Proteins/genetics; Bacterial Proteins/genetics; Bacterial Proteins/metabolism; Base Sequence; Coronavirus/genetics; Coronavirus/immunology; DNA Mutational Analysis; Epitopes/analysis; Escherichia coli/genetics; Escherichia coli Proteins; Fimbriae, Bacterial/genetics; Gene Expression Regulation, Bacterial/genetics; Membrane Glycoproteins/genetics; Membrane Glycoproteins/immunology; Molecular Sequence Data; Mutagenesis, Insertional; Plasmids/genetics; Protein Processing, Post-Translational; Protein Sorting Signals/genetics; Protein Sorting Signals/metabolism; Recombinant Fusion Proteins/biosynthesis; Swine; Viral Envelope Proteins/genetics; Viral Envelope Proteins/immunology |
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