ADVANTAGES

Much more than phage or other cellular display

A universal display system must have a number of crucial features. It should pose minimal restrictions on the size and structure of the protein to be displayed. The system should utilize standard protocols in molecular biology and standard bacterial strains. Tools must also be available to analyze and evaluate the efficiency of passenger display. Optimally, the system should be simple and easy to handle to minimize unexpected obstacles.

The autodisplay system has been developed to fulfill these requirements to the greatest possible extent and has a number of advantages over other display technologies. Especially highly complex structures can be built up with more than 100,000 molecules per cell. The proteins on the surface are immobilized and stabilized, fully accessible and the multimerisation/aggregation of protein complexes is possible.

Furthermore, the purification is very easy, the DNA is an internal label and there is self-replication. As each E. coli could have a different protein on its surface, the specific binding of corresponding external proteins is possible. The target structures can be selected from the mixture by efficient analytical methods like fluorescence activated cell sorting (FACS).

The autodisplay system has some striking advantages in comparison to the other cell surface display systems described to date.

Advantage 1: Large number of reccombinant proteins displayed on the surface of E. coli

Advantage 2: Mobility of the β-barrel serving as an anchor for the recombinant protein within the outer membrane

Advantage 3: Possibility of incorporating an inorganic prosthetic group into an apoprotein expressed at the cell
                       surface of E. coli

Advantage 4: Surface expression of large numbers of recombinant proteins does not reduce cell viability or integrity

Advantage 1: Large number of recombinant proteins displayed on the surface of E. coli

The first advantage is the large number of recombinant proteins displayed on the surface of E. coli. The expression level of the recombinant protein was identical to or even exceeded that of OmpA, which is known to be expressed very constantly with 200 000 molecules per E. coli cell. This was significantly more than has been reported for other surface-display systems like the Lpp-OmpA hybrid system in E. coli and the fibronectin binding protein based system in S. carnosus.

Advantage 2: Mobility of the β-barrel serving as an anchor for the recombinant protein within the outer membrane

A second striking advantage of the autodisplay system is the mobility of the β-barrel serving as an anchor for the recombinant protein within the outer membrane. This enables passenger proteins, which are expressed as monomers within the context of the autodisplay system, to form functional dimers at the cell surface. Such a passenger-driven, spontaneous dimerization of surface-exposed domains has not yet been reported for any other cell surface display system investigated. Passenger-driven dimerization offers interesting prospects for future applications of the autodisplay system. The system could also be used for the surface expression of multihomomeric proteins, as was shown for the tetradecameric nitrilase from Alcaligenes faecalis. Even the display of heterotetrameric enzymes like human CK2 (2α/2β-subunits) was possible. These results indicate that the autodisplay system is a convenient tool for the display of a broad range of different enzymes, making them accessible for biocatalysis and bioanalytics.

Advantage 3: Possibility of incorporating an inorganic prosthetic group into an apoprotein expressed at the cell surface of E. coli

Another outstanding characteristic of the autodisplay system is the possibility of incorporating an inorganic prosthetic group into an apoprotein expressed at the cell surface of E. coli. The proof of principle was established as bovine adrenodoxin (Adx), a [2Fe-2S] cluster carrying electron transfer protein, was displayed in an active state. Using autodisplay it is also possible to display proteins containing cofactors, such as heme containing bacterial and human P450 enzymes or flavin adenine dinucleotide (FAD) carrying NADH-oxidase.from Lactobacillus brevis.

Advantage 4: Surface expression of large numbers of recombinant proteins does not reduce cell viability or integrity

Finally, the most convenient feature of autodisplay is that surface expression of large numbers of recombinant proteins does not reduce cell viability or integrity. Cells survived challenging analytic approaches, including fluorescence activated cell sorting (FACS). FACS enables the selection of single cells having a specific feature from a library of variants and clonal expansion for sequence determination or structural analysis of the molecule displayed at the cell surface. This strategy was successfully applied for the identification of new cathepsin G inhibitors from random peptide libraries and is a first indication that the high number of molecules displayed on the surface of a single cell combined with persistent cell integrity could afford direct selection of cells displaying the correct molecules from a library instead of requiring enrichment or panning positive cells.