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-termina l domains promoting the translocation of the N-terminally attached pas senger domains across gram-negative bacterial outer membranes. Compute r predictions identified the C-terminal domain of the Escherichia coli adhesin involved in diffuse adherence (AIDA-I) as a member of the aut otransporter family. A model of the P-barrel structure, proposed to be responsible for outer membrane translocation, served as a basis for t he construction of fusion proteins containing heterologous passengers. Autotransporter-mediated surface display (autodisplay) was investigat ed for the cholera toxin B subunit and the peptide antigen tag PEYFK. Up to 5% of total cellular protein was detectable in the outer membran e as passenger autotransporter fusion protein synthesized under contro l of the constitutive P-TK promoter. Efficient presentation of the pas senger domains was demonstrated in the outer membrane protease T-defic ient (ompT) strain E. coli UT5600 and the ompT dsbA double mutant JK32 1. Surface exposure was ascertained by enzyme-linked immunosorbent ass ay, immunofluorescence microscopy, and immunogold electron microscopy using antisera specific for the passenger domains. In strain UT2300 (o mpT(+)), the passenger domains were released from the cell surface by the OmpT protease at a novel specific cleavage site, R down arrow V. A utodisplay represents a useful tool for future protein translocation s tudies with interesting biotechnological possibilities.