Autodisplay: one-component system for efficient surface display and release of soluble recombinant proteins from Escherichia coli.
The immunoglobulin A protease family of secreted proteins are derived from self-translocating polyprotein precursors which contain C-terminal domains promoting the translocation of the N-terminally attached passenger domains across gram-negative bacterial outer membranes. Computer predictions identified the C-terminal domain of the Escherichia coli adhesin involved in diffuse adherence (AIDA-I) as a member of the autotransporter family. A model of the beta-barrel structure, proposed to be responsible for outer membrane translocation, served as a basis for the construction of fusion proteins containing heterologous passengers. Autotransporter-mediated surface display (autodisplay) was investigated for the cholera toxin B subunit and the peptide antigen tag PEYFK. Up to 5% of total cellular protein was detectable in the outer membrane as passenger autotransporter fusion protein synthesized under control of the constitutive P(TK) promoter. Efficient presentation of the passenger domains was demonstrated in the outer membrane protease T-deficient (ompT) strain E. coli UT5600 and the ompT dsbA double mutant JK321. Surface exposure was ascertained by enzyme-linked immunosorbent assay, immunofluorescence microscopy, and immunogold electron microscopy using antisera specific for the passenger domains. In strain UT2300 (ompT+), the passenger domains were released from the cell surface by the OmpT protease at a novel specific cleavage site, R / V. Autodisplay represents a useful tool for future protein translocation studies with interesting biotechnological possibilities.
Maurer
J
J
Abteilung Infektionsbiologie, Max-Planck-Institut für Biologie, Tübingen, Germany.
Jose
J
J
Meyer
T F
TF
D018830:Adhesins, Escherichia coli / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D000595:Amino Acid Sequence; D000941:Antigens / Q000235:genetics; D001483:Base Sequence; D001692:Biological Transport / Q000235:genetics; D002352:Carrier Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D002462:Cell Membrane / Q000378:metabolism; D002772:Cholera Toxin / Q000096:biosynthesis* / Q000235:genetics; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics*; D005455:Fluorescent Antibody Technique; D005818:Genetic Engineering / Q000379:methods*; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D016253:Microscopy, Immunoelectron; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry; D017421:Sequence Analysis; D012697:Serine Endopeptidases / Q000378:metabolism; D012995:Solubility
C075559:AIDA-I protein, E coli; D018830:Adhesins, Escherichia coli; D000941:Antigens; D002352:Carrier Proteins; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D002772:Cholera Toxin; D012697:Serine Endopeptidases; C057840:omptin outer membrane protease