Fluorescently Labeled Nanobodies

In 1993 Hamers and co-workers discovered antibodies in the camel serum that have naturally evolved to be devoid of light chains^[185]. Later, these very small single-domain antibodies, about 15 kDa^[186], were shown to be adaptable to other desired antigens by immunizing dromedaries followed by a screening process that led to extremely stable, highly soluble minimum sized antigen binders. These binders, termed nanobodies (Nb) by the Ablynx company, could be recombinantly expressed in E. coli and were proven to react specifically and with high affinity to the antigens^[187].

Due to their advantageous properties nanobodies have been used in a broad range of applications as research and diagnostic tools as well as therapeutics^[188]. Saerens et al.

found Nbs to sense conformational changes on different prostate-specific antigen isoforms which could be helpful to discriminate the stages of prostate cancer^[189]. Nanobodies served as tools for molecular tumor imaging^[190,191] and were further developed for in vivo imaging of specific immune cell types^[186]. The Leonhardt laboratory engineered an anti-GFP

nanobody that allows the purification of GFP-fusion proteins and their interacting partners for biochemical studies on, e.g., DNA binding, enzymatic activity and complex formation^[192]. The Rizzoli group (ENI, Göttingen) is interested in synaptic vesicle function, with an emphasis on synaptic vesicle recycling, and therefore combines fluorescently labeled nanobodies and high resolution imaging provided by stimulated emission depletion (STED^[193,194]) microscopy. Chemical ligations of fluorophores and Nbs prevent a quantitative analysis due to multiple but erratic binding. Here, we show a collaborative approach for the site-specific incorporation of fluorophores, suitable for STED^[125], by applying the principles of genetic code expansion as described in Ch. 5.2.

First, a set of α-synuclein targeting Nbs^[195] (NbSyn2), including wild type and the amber codon containing mutants S44TAG, K70TAG, R72TAG, K93TAG, K115TAG and S153TAG (provided by Felipe Opazo from AG Rizzoli),were expressed in the absence and presence of BocK and NorK to test the overall incorporation of UAAs into nanobodies (Figure 5.7).

Figure 5.7: Incorporation of BocK and NorK into NbSyn2.

A) Expression of wild type (WT) and amber codon containing nanobodies NbSyn2 (pCLA175 to pCLA181) in E. coli BL21 was induced with IPTG (1 mM final concentration) after two hours of incubation at 37 °C. BocK (1 mM final concentration) was added subsequently and temperature was shifted to 28 °C for overnight expression (∼16 h). PylS and PylT were used as aaRS and tRNA pair (pCLA97). For the western blot whole cell extracts were separated with SDS PAGE (Ch. 3.2.2.3) and blotted onto a PVDF membrane (Ch. 3.2.2.4). Anti-His-antibody was used as primary Anti-His-antibody. B) Same experiment as in A) but NorK was used as UAA instead of BocK together with the appropriate aaRS AzpcKRS (pCLA166). WT and R72 samples on the right side originate from A).

We found UAA dependent expression of NbSyn2 with BocK being incorporated with higher efficiencies than NorK yielding more full-length protein, as seen before (Figure 5.4-B). The clones with amber codons at positions K70 and R72 revealed the best expression levels.

Both mutations are located in the same loop opposite the target binding pocket, according to the crystal structure of the nanobody (pdb file 2X6M^[195], not shown).

We finally chose the NbSyn2 K70TAG clone for large scale expression and subsequent labeling with the Abberior Star635 fluorophore. Since the nanobody was cloned behind a secretion signal its production occurred in the periplasm, where the oxidizing environment is optimal for the required disulfide formation^[188]. Secreted proteins were purified from the medium (1 L; Ch. 3.2.2.8) yielding 0.11 mg full-length protein and then labeled with the tetrazine conjugated fluorophore (Ch. 3.2.2.11). Labeling was verified and visualized using a Typhoon imager (Figure 5.8).

Figure 5.8: Fluorescently labeled NbSyn2.

NorK was incorporated into nanobody NbSyn2 (K70TAG; pCLA177). NbSyn2-NorK was labeled with the fluorophore Abberior Star635 (Abb. 635) conjugated with a tetrazine. The SDS-PAGE gel was scanned using a Typhoon imager (Excitation: 633 nm (red laser) and emission: Filter 670 nm BP30).

NbSyn2 could be successfully labeled with the tetrazine conjugated fluorophore Abberior Star635 and was given to Felipe Opazo for the removal of unbound dye and further experiments.

Due to the low yield of purified NbSyn2 from medium, we tested a second nanobody targeting GFP ^[192] (NbGFP). NbGFP is expressed in good yields and exhibits excellent binding to its target. Additionally, the cell line was changed from E. coli BL21 to E. coli SHuffle.

SHuffle cells were engineered to assist disulfide bond formation in the cytoplasm, e.g., in nanobodies, rendering the transport of proteins into the periplasm unnecessary (see NbSyn2). This is achieved by the simultaneous deletion of the reductases trxB and gor and the constitutive expression of a chromosomal copy of the disulfide bond isomerase DsbC which promotes the correction of mis-oxidized proteins into their correct form^[196–198]. This cell line also possesses a disadvantage in that a chromosomal copy of the resistance gene for spectinomycin, which is also present on the pCDF plasmids used for the modular genetic tools.

To this end, we cloned the resistance genes for chloramphenicol and ampicillin from the Duet vectors pACYC and pET, respectively, into the modular genetic tools pCLA116 and pCLA171, replacing the spectinomycin resistance genes (pCLA184 to pCLA187).

In order to test the genetic code expansion system in the SHuffle cell line, we prepared competent cells harboring the optimized modular genetic tool with an ampicillin resistance gene and encoding BocK in response to an amber codon (pCLA185). These cells were transformed with plasmids for NbGFP containing amber codons at positions Q13 and R76 (pCLA189 and pCLA192; provided by Felipe Opazo from AG Rizzoli). Nanobodies were expressed in the absence and presence of BocK (Figure 5.9).

Figure 5.9: Incorporation of BocK into NbGFP.

Expression of amber codon containing nanobodies NbGFP (pCLA189 and pCLA192) in E. coli SHuffle was induced with IPTG (1 mM final concentration) after 2.5 hours of incubation at 30 °C. BocK (1 mM final concentration) was added subsequently and temperature was shifted to 28 °C for overnight expression (∼16 h). PylS and PylT were used as aaRS and tRNA pair (pCLA185). Control cells (ctrl) contained only pCLA185 but no NbGFP plasmid.

For the western blot whole cell extracts were separated with SDS PAGE (Ch. 3.2.2.3) and blotted onto a PVDF membrane (Ch. 3.2.2.4). Anti-Myc-antibody was used as primary antibody.

The adapted modular genetic tool containing the ampicillin resistance gene allowed the incorporation of the UAA BocK into NbGFP. We found UAA dependent expression with clone R76TAG showing more full-length protein. Since the Myc-tag is cloned to the C-terminus of the protein only full-length nanobodies should be detected by the antibody.

Therefore, the intense signals of smaller sizes are probably due to strong degradation.

In summary, we were able to encode UAAs in both nanobody proteins, NbSyn2 and NbGFP.

NbSyn2 was successfully labeled with a fluorophore suitable for STED, opening an avenue for highly specific binding to its target followed by quantifiable high resolution microscopy.

First experiments with NbGFP expression in E. coli SHuffle cells looked promising concerning high yield expression. The use of this cell line will simplify the purification of the nanobodies enormously.