Team:Penn/SurfaceDisplay

Having demonstrated that our INPNC surface display system could localize a desired protein (mCherry) to the outer membrane surface (), we sought to display the picomolar affinity, HER2 binding Designed Ankyrin Repeat Protein (DARPin) H10-2-G3 on the surface of our BL21 cells. This would allow for bacterial targeting to HER2 over-expressing cells, that could then be lysed in a spatially accurate manner using our light-activated ClyA expression system. Such surface display of a large antibody-mimetic protein is unprecedented, and assaying whether it had actually occurred would be difficult, because unlike mCherry, DARPin has no native fluorescence. We also did not have the microscopy resources to resolve the membrane from the cytoplasm, or flow cytometry systems with high enough resolution for bacteria.

To achieve and assay DARPin surface display, we constructed a C-terminal INPNC-DARPin fusion in the same manner as our INPNC-mCherry fusion (using a 12aa GS Linker), except we added a Human Influenza Aggregation (HA) tag to the N-terminal of DARPin to allow us to use antibodies to assay whether it had been localized to the membrane. After expression and 48-hour 0.3mM IPTG induction of INPNC-DARPin-HA in BL21 cells using the pET26b expression vector, we immunostained cells using an anti-HA, Alexa-fluor 647 conjugated antibody (Cell Signaling Technologies). Since antibodies are not permeable to the E. coli membrane, after washing the cells there should be signal only on those cells that have displayed the HA tag on their surface.

We then took a small amount of HA immunostained bacteria and visualized them by confocal microscopy at 630x magnification using a 633nm He-Ne laser. Using this immunostaining visualization method, we were able to demonstrate for the first time that DARPin H10-2-G3 had in fact been localized to the cell surface (Figures 1-8).

We first stained control bacteria expressing DARPin-HA without fusion to INPNC (Figures 1-2). The following images were taken of identical densities of bacteria as verified by phase contrast. The lack of fluorescence in the induced condition indicates that the antibody was not permeable to the bacterial membrane, because it did not bind to the cytoplasmic DARPin-HA.

Figure 1

Figure 1: Uninduced pET26b-DARPin-HA (-IPTG) bacteria are not immunostained by anti-HA antibody.

Figure 2

Figure 2: Induced pET26b-DARPin-HA (+IPTG) bacteria are not immunostained by anti-HA antibody.

Then, to determine whether INPNC could display an HA tag on the surface of E. coli, we stained bacteria expressing INPNC-HA and found that IPTG-induced bacteria exhibited strong 647nm fluorescence when excited by the 633nm laser (Figures 3-4). This indicated that the C terminus of INPNC is displayed on the outer membrane as expected.

Figure 3

Figure 3: Uninduced pET26b-INPNC-HA (-IPTG) bacteria are not immunostained by anti-HA antibody.

Figure 4

Figure 4: Induced pET26b-INPNC-HA (+IPTG) bacteria are strongly immunostained by anti-HA antibody, indicating surface expression.

Finally, to determine whether we could display our HA-tagged DARPin on the surface of E. coli, we stained INPNC-DARPin-HA expressing bacteria and found strong immunofluorescence under the induced condition (Figures 5-6), indicating that DARPin-HA had been displayed on the outer membrane.

Figure 5

Figure 5: Uninduced pET26b-INPNC-DARPin-HA (-IPTG) bacteria are not immunostained by anti-HA antibody

Figure 6

Figure 6: Induced pET26b-INPNC-DARPin-HA (+IPTG) bacteria are strongly immunostained by anti-HA antibody, indicating surface expression.

We also repeated the experiment without the antibody to rule out any possibility of autofluorescence of DARPin-HA. As expected, no fluorescence was detected in the induced and uninduced conditions when INPNC-DARPin-HA bacteria were not stained with the anti-HA Alexa-Fluor 647 antibody (Figures 7-8).

Figure 7

Figure 7: Uninduced pET26b-INPNC-DARPin-HA (-IPTG) (-AB) bacteria are not fluorescent.

Figure 8

Figure 8: Induced pET26b-INPNC-DARPin-HA (-IPTG) (-AB) bacteria are not fluorescent.

Since we detected outer membrane fluorescence of INPNC-DARPin-HA, all negative and positive controls performed as expected, and the same display system also worked with mCherry, we are confident that DARPin H10-2-G3 has been displayed on the surface of E. coli for the first time. This staining was repeated on two other occasions and the same results were determined.