Chromophore pre-maturation for improved speed and sensitivity of split-GFP monitoring of protein secretion

Complementation-dependent fluorescence is a powerful way to study co-localization or interactions between biomolecules. A split-GFP variant, involving the self-associating GFP 1–10 and GFP 11, has previously provided a convenient approach to measure recombinant protein titers in cell supernatants. A limitation of this approach is the slow chromophore formation after complementation. Here, we alleviate this lag in signal generation by allowing the GFP 1–10 chromophore to mature on a solid support containing GFP 11 before applying GFP 1–10 in analyses. The pre-maturated GFP 1–10 provided up to 150-fold faster signal generation compared to the non-maturated version. Moreover, pre-maturated GFP 1–10 significantly improved the ability of discriminating between Chinese hamster ovary (CHO) cell lines secreting GFP 11-tagged erythropoietin protein at varying rates. Its improved kinetics make the pre-maturated GFP 1–10 a suitable reporter molecule for cell biology research in general, especially for ranking individual cell lines based on secretion rates of recombinant proteins.

Supplementary figure S2 -Evaluation of incubation temperature and time for GFP 1-10 chromophore pre-maturation on His6-Z_GFP 11-coupled beads Figure S2. Evaluation of incubation temperature and time for GFP 1-10 chromophore pre-maturation on His6-Z_GFP 11-coupled beads. Approximately 100 µl bead slurry of His6-Z_GFP 11-coupled beads were mixed with 1 mL of 2 mM GFP 1-10 in 1.5 ml tubes. The maturation protocol was carried out for five hours, overnight, or for 72 hours and at 4°C or room temperature as indicated. After acid release and pH adjustment of GFP 1-10 mat , His6-Z_GFP 11 protein was added for monitoring of the fluorescence complementation. The highest fluorescence was achieved for maturation for 72 hours at room temperature. However, there was only a slight difference seen between 72 hours and overnight incubation at RT; hence, the quicker overnight protocol was selected for future pre-maturations. Approximately 100 µl bead slurry of His6-Z_GFP 11-coupled beads were mixed with 1 ml of 2 mM GFP 1-10 in 1.5 ml tubes. The pre-maturation was carried out at room temperature overnight in transparent plastic tubes, either covered in aluminum foil (dark conditions) or left uncovered in illuminated laboratory space (light conditions). His6-Z_GFP 11 was subsequently added to acid-released and pH-adjusted GFP 1-10 mat .
No apparent difference in the fluorescence development kinetics was seen between the GFP 1-10 mat that had been exposed to light during the pre-maturation from that not exposed to light. Fluoresence intensity (a.u.)

Time (min)
Overnight -RT -Dark Overnight -RT -Light Supplementary figure S4 -Effects of bead-to-His6-Z_GFP 11 ratio on GFP 1-10 pre-maturation Figure S4. Effects of bead-to-His6-Z_GFP 11 ratio on GFP 1-10 pre-maturation. (a) 2.5 ml of 0.87 mM, 0.44 mM or 0.087 mM solutions of His6-Z_GFP 11 were coupled to 5 ml of NHS-activated bead slurry. 40 ml of 50 µM GFP 1-10 was subsequently added to the beads for pre-maturation overnight at room temperature. After incubation, beads were washed three times in TNG buffer and eluted with 0.1 M glycin (pH 2.0). The eluate was neutralized to pH 7.4 by addition of an equal volume of 0.5 M Tris-HCl buffer (pH 7.8). The buffer was subsequently exchanged to TNG on a PD-10 desalting column. The fluorescence of the GFP 1-10 mat was measured in duplicates (one replicate for 0.087 mM sample) after the addition of 5 times molar excess of His6-Z_GFP 11 protein.
The amount of GFP 1-10 mat increased with higher densities of His6-Z-GFP 11 on the beads. (b) However, a decrease in the specific yield (obtained fluorescence signal per mg of His6-Z-GFP 11 on beads) was seen for beads immobilized with 0.87 mM compared to 0.44 mM, presumably due to sterical hindrance. Figure S5. Coupling of His6-Z-GFP 11 to NHS beads. To investigate the capacity of the NHS-activated beads, the highest concentration investigated (2.5 ml of 0.087 mM) of His6-Z-GFP 11 was coupled to 5 ml of NHS-activated bead slurry according to the manufacturer's protocol. The amounts of ligand in the supernatant from before (1 µl sample) and after (5 µl sample) coupling was analyzed by SDS-PAGE and Coomassie staining. The results showed that essentially all of the applied His6-Z-GFP 11 protein had been immobilized on the beads from the 0.87 mM His6-Z-GFP 11 solution.

Supplementary figure S6 -Concentration comparison of GFP 1-10 and GFP 1-10 mat by SDS-PAGE Figure S6. Concentration comparison of GFP 1-10 and GFP 1-10 mat by SDS-PAGE.
The concentration in solutions of the two versions were compared on a Coomassiestained SDS-PAGE gel to investigate whether the samples had the same amount of GFP 1-10. The concentration determination by absorbance at 280 nm used to obtain equal amounts for fluorescence assays could potentially have been misleading if GFP 1-10 had more impurities compared to GFP 1-10 mat . Hence, The same theoretical amounts (1 µg) of GFP 1-10 and GFP 1-10 mat (according to A280 concentration determination) were loaded on the SDS-PAGE gel. No clear difference in concentration was seen for the GFP 1-10 solutions used in the complementation assays.
Supplementary figure S7 -Raw data plot for comparison of the kinetics of fluorescent signal generation using GFP 1-10 mat and GFP 1-10 Figure S7: Raw data plot for comparison of the kinetics of fluorescent signal generation using GFP 1-10 mat and GFP 1-10. GFP 1-10 mat and GFP 1-10 were compared head-to-head in TNG buffer containing His6-Z-GFP 11. This figure presents the same data as Figure 3; here, the negative control have not been subtracted from the actual samples to demonstrate the autofluorescence of GFP 1-10 mat . Three replicates were used for both actual samples and negative controls. A plot with only non-maturated GFP 1-10 can be found below.
Supplementary figure S9 -Emission and excitation spectra for GFP 1-10 and GFP 1-10 mat Fig. S9. Emission and excitation spectra for GFP 1-10 mat and GFP 1-10. Excitation and emission spectra were measured on a CLARIOstar® plate reader (BMG Labtech) with emission set to 545 nm (±8 nm) for recording of excitation spectra and with excitation set to 466 nm (±8 nm) for recording of emission spectra. Measurements were conducted with i) 4 500 pmol GFP 1-10 and 4700 pmol His6-Z_GFP 11, (ii) 4500 pmol GFP 1-10 alone, (iii) 94 pmol GFP 1-10 mat and 4700 pmol His6-Z_GFP 11 and (iv) 94 pmol GFP 1-10 mat alone in TNG buffer and a total volume of 100 µl. The large amount of non-maturated GFP 1-10 used in the assays was necessary to get readable signals. Excitation and emission measurements were done at various time points after addition of His6-Z_GFP 11 but the here presented excitation and emission spectra were generated after 90 minutes and 2 hours, respectively. Measurements were also performed at 488 nm excitation and 520 nm emission to evaluate the signal at the maximum emission and excitation wavelengths (data not shown). These results showed the same excitation and emission patterns for both GFP 1-10 and GFP 1-10 mat , but due to limitation of overlapping signals, they did not show the full spectral view of interest for each protein. The results showed that the