Creation of targets for proteolytic cleavage in the LamB protein of E coli K12 by genetic insertion of foreign sequences: implications for topological studies.
LamB, an integral outer membrane protein of E coli K12, is highly resistant to protease digestion. We had previously genetically inserted a foreign sequence corresponding to an epitope from the poliovirus next to amino acids 146, 153, 189, and 374 of LamB. In 3 cases (sites 146, 153, 374), insertion of the foreign peptide did not extensively affect the functions of LamB (and therefore folding). In 2 cases (sites 146 and 374) the polio virus epitope was detectable on the bacterial surface with a specific monoclonal antibody. We show here that the 4 modified proteins are sensitive to trypsin, including on intact cells. The sizes of the major cleavage products is that expected for proteolysis at or near the sequences inserted. In 1 case (site 153), this was directly demonstrated by protein sequencing. The results confirm the cell surface exposure of the regions of residues 153 and 374 and provide information on the regions around residues 146 and 189. Perspectives and limitations of this approach for fine studies on the mode of insertion of membrane proteins are briefly discussed.
Ronco
J
J
Unité de Programmation Moléculaire et Toxicologie Génétique, CNRS UA271 INSERM U163, Institut Pasteur, Paris, France.
Charbit
A
A
Hofnung
M
M
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology; D000914:Antibodies, Viral / Q000276:immunology; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D017955:Poliovirus / Q000235:genetics / Q000276:immunology; D018272:Porins; D011487:Protein Conformation; D015202:Protein Engineering; D011991:Receptors, Virus / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000276:immunology; D013379:Substrate Specificity; D014357:Trypsin / Q000378:metabolism; D014764:Viral Proteins / Q000235:genetics
D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D001425:Bacterial Outer Membrane Proteins; D010446:Peptide Fragments; D018272:Porins; D011991:Receptors, Virus; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; C016532:maltoporins; D014357:Trypsin