Antibodies against synthetic peptides and the topology of LamB, an outer membrane protein from Escherichia coli K12.
LamB, an outer membrane protein from Escherichia coli K12, is involved in the transport of maltose and maltodextrins across the outer membrane and constitutes a receptor for a number of bacteriophages. A recent folding model proposes that LamB spans the outer membrane through a number of transmembranous segments separated by regions exposed either to the cell exterior or to the periplasm. This model is essentially based on predictions of structure and genetic arguments relying on the hypothesis that the mutations studied did not alter the folding of the protein. In order to obtain direct evidence with the unaltered protein, we elicited polyclonal antibodies against synthetic peptides corresponding to several LamB sequences. We chose four regions. Three of them [aa 147-161 (peptide 2), aa 371-385 (peptide 3), and aa 399-413 (peptide 4)] are predicted to face the outside of the cell, and the fourth (aa 19-33 (peptide 1)] is predicted to be periplasmic. By immunoblotting against extracts of various mutants, these antibodies were shown to be specific for LamB and targeted to the selected regions. In some cases, the recognition sites for antibodies were narrowed down to parts of a region. In vivo, on intact cells, anti-peptides 2, 3, and 4 reacted with LamB in an ELISA; this confirmed that regions of peptide 2 and 3 are located, at least in part, at the cell exterior and provided the first proof for a similar, situation of the region of peptide 4. Under the same conditions, anti-peptide 1 did not react with LamB.(ABSTRACT TRUNCATED AT 250 WORDS)
Molla
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Unité de Programmation Moléculaire et Toxicologie Génétique (CNRS UA 271-INSERM U163), Paris, France.
Charbit
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Le Guern
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Ryter
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Hofnung
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D000595:Amino Acid Sequence; D000906:Antibodies / Q000302:isolation & purification; D000918:Antibody Specificity; D001425:Bacterial Outer Membrane Proteins / Q000032:analysis / Q000235:genetics* / Q000276:immunology; D010582:Bacteriophage lambda / Q000235:genetics; D002352:Carrier Proteins / Q000032:analysis; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000032:analysis / Q000235:genetics*; D015151:Immunoblotting; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D010455:Peptides / Q000276:immunology*; D010957:Plasmids; D018272:Porins; D011487:Protein Conformation; D011991:Receptors, Virus / Q000032:analysis / Q000235:genetics* / Q000276:immunology
D000906:Antibodies; D001425:Bacterial Outer Membrane Proteins; D002352:Carrier Proteins; D010455:Peptides; D018272:Porins; D011991:Receptors, Virus; C016532:maltoporins