Exogenous gene transfer into the brain is an important strategy to study in vivo protein function, investigate pathogenic mechanisms of diseases as well as develop new therapeutic strategies. However, the study of exogenously expressed proteins requires rigorous assessment of transgene expression, preferentially distinct from the corresponding endogenous protein. In this study, we have used lentiviral vector-based expression of small epitope tags (AU1, flag, 3flag, myc, V5, HA) N-terminally fused to eGFP in rat brain to evaluate immunohistochemical detection of several commonly used epitope tags in floating sections. In this way, we have evaluated the performance of several commonly used epitope tag/antibody combinations.
First, we evaluated the detection of the different epitope tags in HEK293T cell extracts after transduction with lentiviral vectors encoding the different tag-eGFP fusion proteins. Western blot analysis shows a distinct and specific detection using our in-house anti-eGFP antibody as well as all examined anti-tag antibodies. Especially the V5/anti-V5 antibody and 3flag/FlagM2 antibody combinations allow a very sensitive detection in cell extracts (Fig. 1C).
The main goal of this study was to compare in vivo detection of different frequently used epitope tags using commercially available antibodies. Chromogenic DAB-based as well as fluorescent immunohistochemical detection of the overexpressed tags revealed that all epitope tag/antibody combinations, with the exception of myc/polyclonal anti-myc, meet the requirements of a specific and sensitive detection in rat brain (Fig. 2, 3 and 4). The specificity of the V5/anti-V5 and HA/HA11 combinations are comparable with the other combinations; however the sensitivity seems to be higher since lower antibody concentrations were used for optimal immunohistochemical detection (Fig. 3 and Table 2). These differences were most apparent for the immunohistochemical detection with DAB. The polyclonal anti-myc antibody on the other hand, detects its antigen with low sensitivity as well as low specificity. Therefore, using the presented protocol, our findings show that all tag/antibody combinations, with the exception of myc/polyclonal anti-myc, show sufficient sensitivity and specificity for immunohistochemical analysis in brain tissue.
Based on the strong increase in sensitivity in western blotting detection, we expected the detection of the 3flag tag in vivo to be more sensitive compared to the flag tag; however no significant differences in sensitivity were observed between these two tags using either the monoclonal FlagM2 antibody or a polyclonal anti-flag antibody.
In terms of sensitivity, our in-house eGFP antibody was superior to all tested tag antibodies. However by using a large protein such as eGFP itself as tag for a protein of interest, caution should be exercised since eGFP fusion might interfere with the normal protein function, especially for small proteins. An alternative strategy could consist of a bicistronic construct using a T2A-like peptide , although a minor fraction of fusion protein might be produced with this system.
As proof-of-principle experiment, we overexpressed firefly luciferase tagged with V5 and co-expressed with eGFP in mouse striatum. Luciferase activity was confirmed via bioluminescence imaging. The enzymatic activity of fLuc was not affected by tagging with V5 since the BLI signal per injected transducing unit vector does not differ from untagged fLuc (3,3 p/s/TU for V5-fLuc versus 2,3 p/s/TU for untagged fLuc). However, immunohistochemical analysis using a commercial anti-luciferase antibody failed to detect the transgene. In contrast, we were able to clearly detect the V5-luciferase using the V5 antibody (Fig. 5). The detection sensitivity with the V5 antibody appears somewhat lower than in the case of the V5-eGFP fusion protein (Fig. 2). This may be attributed to differences in stability of the two proteins (eGFPT1/2: >10 h, fLucT1/2: 3 h) since initial protein levels of V5-luciferase and eGFP are identical when co-expressed using a T2A sequence . This example clearly underlines the advantage of epitope tagging for sensitive and specific detection of an exogenous protein which is otherwise not readily detectable with IHC.
In order to confirm the previous data with another tag, 3flag-luciferase-T2A-eGFP was injected and BLI signals were measured. We could confirm that the activity of tagged fLuc is maintained and even slightly increased (157,20 p/s/TU for 3flag-fLuc versus 38,84 p/s/TU for untagged fLuc). We therefore conclude that tagging of luciferase with either a V5 or 3flag tag does not adversely affect sensitivity of bioluminescence imaging in the brain.
One of the main concerns fusing a small epitope tag to a protein of interest is the potential impact on properties such as biodistribution, stability, enzymatic activity and binding capacity. Since such effects cannot be predicted, control studies comparing the tagged protein with the wild-type counterpart should always be included [13–15]. However, interference with the tertiary structure and bioactivity can be minimized by using short peptide sequences fused to the terminus of the protein .
Besides immunohistochemical detection evaluated in the present study, fusing a small peptide to any gene of interest also offers great benefit in several other applications. For instance, epitope tags can be used as an affinity tag to perform protein purification when using stringent purification conditions or to identify interacting proteins when combining non-stringent affinity purifications and mass spectrometry analysis of co-purified proteins [16–27]. Moreover, modifications of the small peptide sequence may further enlarge the range of applications . An interesting new development is for example the generation of a drug-controllable tag .
It should be noted that since the choice of tag/antibody combinations is endless and the ideal epitope tag may vary for every transgene and every application, there is still a need for optimization and characterization studies when tagging a new protein [17, 30]. The evaluation of several commonly used tag/antibody combinations in vivo presented here can be used as an aid in the selection of tags to assess in priority. Moreover, we offer a straightforward protocol for immunohistochemical analysis of tagged exogenous protein expressed in rodent brain which can be readily applied for any new protein of interest.