CuAAC reaction

The test CuAAC reaction was performed on cortactin Nb245, 46, 48, 433

, a nanobody that interacts with the cortactin SH3 domain. (For additional information about the test experiment, see 6.2.6 Supplementary information). Alexa Fluor 488 (AF488) was chosen in these test experiments, as a bright and commonly used fluorophore known to be stable for longer time periods and available with both the azide or alkyne reactive moieties. Figure 42A shows the result of the CuAAC reaction when the purified alkyne-nanobody was used directly after the sortase reaction. Because the sortase reaction buffer (50 mM Tris-HCl, 150 mM NaCl, 10 mM CaCl2) contained a high concentration of Cl^- ions, it was possible that the CuAAC reaction was suboptimal⁴²⁴. By changing the buffer to 10 mM HEPES, the reaction proved to be more efficient (Figure 42B), confirming previous observations that a lower concentration or absence of Cl^- ions is preferable for the CuAAC reaction^{434, 435}, since high Cl^- ion concentrations compete for copper, leading to lower efficiency⁴³⁴. Regarding the buffer effect, the range of THPTA

and sodium ascorbate can be adjusted according to⁴³⁶. The experiment was further optimized by comparing the effect of both compounds on the click reaction. The CuAAC reaction was performed once with the initial concentrations (Buffer 1 in Table 2), once with elevated THPTA (Buffer 2 in Table 2) and once with elevated sodium ascorbate (Buffer 3 in Table 2). The addition of sodium ascorbate showed the greatest impact on the CuAAC reaction as shown in Figure 42C. From these experiments, a low concentration of THPTA and increased sodium ascorbate was the best choice. Since sodium ascorbate reduces the Cu(II) to Cu(I), using a higher concentration of sodium ascorbate and lower concentrations of THPTA created a higher risk of hydrolysis due to radicals and/or peroxides as noted in^{437, 438}. The selected concentrations for the CuAAC were set at 0.1 mM CuSO4, 1 mM THTPA and 7.5 mM sodium ascorbate.

Figure 42: Optimization of the labelling of cortactin nanobody 2 - sortag. CuAAC labelling was less efficient when the sortase reaction mixture was used directly for CuAAC (A) compared to dialysis into HEPES buffer before the CuAAC reaction (B). These results were obtained by using the buffer 1 composition (Table 2). Each lane corresponds to 6 µl of the CuAAC reaction mixture. The lowest fluorescent band is free labelled peptide/free fluorophore. (C) Elevated levels of THPTA (Buffer 2 (Table 2)) or Na Ascorbate (Buffer 3 (Table 2)): only an elevated Na Ascorbate concentration showed better labelling efficiency compared to the initial test reaction (Buffer 1 (Table 2)). Each lane corresponds to 6 µl of the CuAAC reaction mixture.

Table 2: Buffer composition overview. Buffer 1-3 were used to determine optimal buffer concentrations for the CuAAC reaction for nanobody labelling.

Buffer 1 Buffer 2 Buffer 3

10 mM HEPES-NaOH 10 mM HEPES-NaOH 10 mM HEPES-NaOH

0.1 mM CuSO4 0.1 mM CuSO4 0.1 mM CuSO4

0.5 mM THPTA 2.5 mM THPTA 0.5 mM THPTA

5 mM sodium ascorbate 5 mM sodium ascorbate 10 mM sodium ascorbate

pH 7.4 pH 7.4 pH 7.4

To monitor the optimal duration of the CuAAC reaction, a sample was taken after 0, 5, 15, 20, 30, 45, 60 and 75 min. As show in Figure 43, the reaction proceeded very rapidly, with a fluorescent band being observed within a few minutes and most of the reaction completed after 1h.

Figure 43: CuAAC reaction time course after incorporation of a para-azido phenylalanine (pAzF). After nanobody production, the nanobodies were dialysed against a HEPES buffer to remove imidazole. This was followed directly by the CuAAC reaction resulting in fluorescently labelled nanobodies. CuAAC reaction time course using cortactin Nb2-pAzF (Nb-pAzF). Coomassie stained gel on the left and corresponding fluorescent signal on the right. The reaction proceeded fast and at 45-60 minutes, saturation was reached. Each lane corresponds to 3 µl of the reaction mixture.

Eluting the nanobodies with the incorporated C-terminal pAzF directly into a compatible buffer can speed up the protocol. The standard buffer used for nanobody purifications^215,

389 contains too high a level of Cl^- ions to be followed by the CuAAC reaction; therefore others used a potassium phosphate buffer⁴³⁴, or preferred a HEPES buffer⁴³⁶. Both are compatible as elution buffers and as buffers for CuAAC^{434, 438}. For the elutions, there is almost no difference using those two buffers as they gave similar yield. When performing CuAAC using these two buffers, similar results were found as shown in Figure 44. As precipitate was observed in some cases after the CuAAC using the potassium phosphate buffer, HEPES was preferred as elution buffer during IMAC purification and as buffer in CuAAC reaction. After CuAAC, an Amicon Ultra-0.5 Centrifugal Filter Unit was used to remove copper ions. This step decreases free AF488, as can be observed in

Figure 44. In a final step, free fluorophore was removed by size exclusion chromatography (SEC) using a PD Spintrap™ G-25.

Figure 44: Purifying Alexa Fluor 488-labeled nanobody. Comparison between CuAAC after using different elution buffers for β-catenin Nb77 (K Phos BFF = potassium phosphate elution buffer, HEPES BFF = HEPES elution buffer). CuAAC was performed for 1h (0h = before CuAAC reaction, 1h = after CuAAC reaction) followed by the removal of copper using an amicon centrifugal filter device (F) and the removal of free AF488 through SEC (PD Spintrap™ G-25). Free AF488 was efficiently removed. (Each lane was loaded with 3 µl of the mixture, making the bands not entirely quantitative due to differences in elution volume.)

To compare the sortase A strategy to the incorporation of pAzF, the quantities of labelled nanobody per L bacterial growth culture is reported in Table 3 and Table 4. The efficiency of each step was similar, but the SrtA method required one additional step (the enzymatic sortase reaction followed by the CuAAC) than the pAzF incorporation method (only CuAAC). The extra step caused a higher reduction in yield compared to the pAzF method.

Table 3: Quantification of purified nanobody obtained per L of bacterial culture via the sortase A labeling method.

(*Cortactin nanobody 2 expresses significantly below average compared to other nanobodies.) Nanobody yield through the sortase A method

Nanobody After production After sortase reaction After CuAAC Cortactin Nb2* 1.7 mg Nb / L culture 0.8 mg Nb / L culture 0.2 mg Nb / L culture

Table 4: Quantification of purified nanobody obtained per L of bacterial culture following pAzF incorporation method.

(*Cortactin nanobody 2 expresses significantly below average compared to other nanobodies.) Nanobody yield through the pAzF incorporation method

Nanobody After production After CuAAC

Cortactin Nb2* 1,4 mg Nb / L culture 0,7 mg Nb / L culture

Β-catenin Nb77 13,9 mg Nb / L culture 9,1 mg Nb / L culture

Β-catenin Nb86 8,9 mg Nb / L culture 5,6 mg Nb / L culture

6.2.3.2 Single step immunocytochemistry with click chemistry labelled