Designed by: Johanna Opgenoorth   Group: iGEM19_Bielefeld-CeBiTec   (2019-10-15)

Proline-glycine-peptide fused to the n-terminus of mCherry
A shot proline-glycine- peptide was fused to the N-terminus of mCherry to investigate the uptake of the fusion protein by A. niger.

Usage and Biology

To investigate processes of endocytosis we fused several S. cerevisiae specific ligands as well as a short proline-glycine-peptide to mCherry. Those fusion proteins enable visualization of the ligand in- and outside the cell. To investigate endocytosis in A. niger we used a virus-like approach by hijacking a proline-transporter to get the peptide into the target cell. Pro_mCherry was characterized together with the three other fusion-proteins Mat_mCherry (BBa_K2926049), Flo_mCherry (BBa_K2926050) and Opy_mCherry (BBa_K2926051).

Sequence and Features

Sequence was validated by Sanger sequencing.

Assembly Compatibility:
  • 10
  • 12
  • 21
  • 23
  • 25
  • 1000

Protein purification

First, the marker protein mCherry (BBa_J06504) was cloned into the expression- and purification-vector pTXB1. To express the desired fusion-proteins the coding sequence of the specific ligands containing a short C-terminal glycine-serine-linker was successfully cloned into the vector pTXB1 coding for mCherry which resulted in four different pTXB1-constructs coding for the fusion-proteins Mat_mCherry, Flo_mCherry and Opy_mCherry. Those fusion-proteins were expressed in E. coli ER2566. The expression was easily detectable, as it was indicated by the red colour of the culture (Fig. 1 and 2).

Fig. 1: Expression culture of the fusion-proteins.
Opy_mCherry, Mat_mCherry, Flo_mCherry and Pro_mCherry (from left to right) were expressed in E. coli ER2566. Expression cultures were cultivated at 37 °C in LB containing 100 mg ampicillin per L to an OD of around 0.6. Expression was induced by addition of IPTG to a final concentration of 0.4 mM. After additional 30 minutes at 37 °C cultures were transferred to 17 °C and protein was expressed over night.

The expression cultures showed different intensities of red which indicated varying levels of expression or a different fluorescence intensity of the expressed proteins.
Fig. 2: Harvested expression culture of the fusion-proteins.
Expression cultures were harvested via centrifugation for 20 min at 4 °C and 4 000 rpm.

After cultivation we compared two different protocols for lysis of the cells. Lysis via Ribolyzer resulted in a much lower yield than lysis via French Press (Fig. 3).
Fig. 3: Ribolyzer (left) and French Press (right).
Harvested cells were lysed using Zirconia metal beads (1 mm) in a Ribolyzer at 8 ;000 rpm for 15 s. Lysis via French Press was performed two times at 16 000 psi with a flow rate of around 1 mL per minute. The lysate was cleared by centrifugation at 4 °C for 1 h and 4 500 rpm.

Purification of the fusion-proteins was performed using the IMPACT-Kit from NEB. The protein of interest had been C-teminally fused to an intein tag that contains a chitin-binding domain. The resulting protein was loaded onto a chitin column (Fig. 4) and washed with a buffer with a high salt concentration.
Fig. 4: Purification columns loaded with the fusion-proteins.
Cleared lysate was loaded onto a chitin column and washed with a buffer with high salt concentration. Finally the protein was eluted, washed in PBS and concentrated.

To cleave the protein of interest from the column it was incubated with DTT for 20-24  hours. After purification the different fusion proteins were analyzed on a SDS-PAGE (Fig. 5).
Fig. 5: SDS-PAGEs of the purification process.
The purification process and the purified proteins were analyzed via SDS-PAGE. E. coli lysate of the expression culture, flow-through- and wash-fraction as well as the purified protein were denatured by heating the samples to 98 °C for 10 min in SDS-PAGE loading buffer containing DTT and loaded on an polyacrylamide-gel (12 %). The proteins were separated through electrophoresis (25 mA). Suggested fusion protein bands in the lane with purified proteins were marked in dark red.

The SDS-PAGE and a subsequent Bradford assay showed that we were able to purify Mat_mCherry with a molecular weight of 28.7 kDa and a yield of 2.35 mg, Opy_mCherry with a molecular weight of 31 kDa and a yield of 1.48 mg, Flo_mCherry with a molecular weight of 48.3 kDa and a yield of 40.9 µg and Pro_mCherry with a molecular weight of 27.7 kDa and a yield of 67.9 µg. To further analyze the expressed fusion-proteins and compare them to the expected protein sequence the marked bands were excised from the SDS-PAGE, washed, digested with trypsine and analyzed in a MALDI-TOF MS (Fig. 6).
Fig. 6: Mass spectrum of the fusion proteins Mat_mCherry (1), Opy_mCherry (2), Flo_mCherry (3) and Pro_mCherry (4) after tryptic digestion compared to the theoretical mass spectrum.
Excised bands from the SDS-PAGEs of Mat_mCherry, Opy_mCherry, Flo_mCherry and Pro_mCherry were washed, digested over night with trypsine and co-cristallyzed with a HCCA-matrix on a MALDI target. Mass spectrum was recorded in a MALDI-ToF MS from Bruker Daltronics and data was evaluated using the software BioTools.

The generated mass spectra and mass lists were evaluated using the software BioTools. To compare the experimentally determined data to the theoretical protein sequence we performed an in silico trypsine-digestion of the expected protein sequence and compared the generated mass spectrum and mass list to the measured ones. We were able to match all four investigated fusion-proteins with the theoretically determined spectra.

Protein characterization

A very important property of the fusion-proteins is the ability to fluoresce independently from the fusion at the N-terminus. To verify this we measured the fluorescence- and absorbance spectra of all four fusion-proteins (Fig. 7).
Fig. 7: Fluorescence- and absorbance-spectra of the fusion proteins.
Emission- (dashed lines) and excitation-spectra (solid lines) of Mat_mCherry (dark red), Opy_mCherry (dark purple), Flo_mCherry (purple) and Pro_mCherry (blue) were measured (λEx=570 nm, λEm=610 nm) using the TECAN infinite M200 and normalized to their maximum.

All four fluorescence spectra look very similar. The absorbance spectra of all four fusion proteins are matching each other very well, too. Overall, the fluorescence- and absorbance-spectra of the fusion-proteins are very similar to the ones measured for mCherry (Fig. 8).
Fig. 8: Fluorescence- and absorbance-spectra of mCherry and Mat_mCherry as an example for a fusion-protein.
Emission- (dashed lines) and excitation-spectra (solid lines) of Mat_mCherry (dark red) and mCherry (grey) were measured (λEx=570 nm, λEm=610 nm) using the TECAN infinite M200 and normalized to their maximum.

To further characterize the fluorescence properties of the purified proteins we diluted the proteins and compared the fluorescence intensity to the one of mCherry standardized to the fluorescence of 0.5 µM Texas Red (Fig. 9)
Fig. 9: Fluorescence intensity of dilution series of the fusion-proteins.
Fluorescence intensity of the dilution series of the fusion-proteins Mat_mCherry (dark red), Opy_mCherry (dark purple), Flo_mCherry (purple), Pro_mCherry (blue) and mCherry (grey) were measured (λEx=570 nm, λEm=610 nm, gain calculated from 2.5 µM Texas Red) using the TECAN infinite M200 and normalized to the fluorescence intensity of 0.5 µM Texas Red at the same wavelength.

As a result we observed, that Pro_mCherry showed the highest fluorescence intensity followed by Flo_mCherry, Mat_mCherry and Opy_mCherry. Compared to mCherry the fluorescence intensity of the fusion-proteins has been lowered (Fig. 10). The fluorescence intensity of 1 µmol Flo mCherry equals the fluorescence of 0.49 µmol Texas Red, the fluorescence intensity of 1 µmol Mat_mCherry equals the intensity of 0.47 µmol Texas Red, the fluorescence intensity of 1 µmol Opy_mCherry equals the intensity of 0.41 µmol Texas Red and the fluorescence intensity of Pro_mCherry equals the fluorescence intensity of 0.54 µmol Texas Red.

Endocytosis assays

Fluorescence in the supernatant

With the purified proteins we performed an endocytosis-assay (Fig. 10). S. cerevisiae was incubated over an hour with 1 µM fusion-protein. Every 15 minutes the fluorescence intensity in the supernatant was determined using a plate reader (Fig. 11).
Fig. 10: Scheme of the carried out endocytosis assay
Target cells are incubated with the fusion-protein. Over the time the cells specifically take up the proteins from the media. This results in a measurable decrease of media-fluorescence.

Fig. 11: Mat_mCherry, Opy_mCherry and mCherry are taken up by S. cerevisiae
S. cerevisiae was incubated in SD media (30 °C, 180 rpm, OD around 0.4, dark) over 1 h with 1 µM mCherry (grey), Mat_mCherry (dark red), Opy_mCherry (dark purple) and Flo_mCherry (purple). Every 15 minutes a sample was taken and the remaining fluorescence in the supernatant was measured using the TECAN infinite M200 (λEx=570 nm, λEm=610 nm, gain calculated from 2.5 µM Texas Red). The results were normalized to the fluorescence intensity at t=0 and the fluorescence intensity of the negative control without cells.

The results show that the fluorescence intensity of Mat_mCherry, Opy_mCherry and mCherry proteins in the supernatant decreases over the time. This indicates, that Opy_mCherry, Mat_mCherry and even mCherry alone seem to interact with S. cerevisiae and might even be taken up by the cell but the specific ligands seem to enhance endocytosis as the faster decrease in media-fluorescence shows. In contrast, the fluorescence intensity of Flo_mCherry in the supernatant did not decrease which led us to the conclusion that it is not taken up by the cell.

The same assay performed for S. cerevisiae was carried out for A. niger to verify the uptake of Pro_mCherry into the cells. Additionally, to investigate the specificity of the tested ligands, A. niger was also incubated with the S. cerevisiae-specific Mat_mCherry (Fig. 12).
Fig. 12: The fusion proteins are selectively taken up by the target A. niger.
A. niger was incubated in SD media (30 °C, 180  rpm, dark) over 1 h with 0.5 µM mCherry (grey), Mat_mCherry (dark red) and Pro_mCherry (blue). After 60 minutes a sample was taken and the remaining fluorescence in the supernatant was measured using the TECAN infinite M200 (λEx=570 nm, λEm=610 nm, gain calculated from 2.5 µM Texas Red). The results were normalized to the fluorescence intensity at t=0 and the fluorescence intensity of the negative control without cells.

Because of the lower growth rate of A. niger compared to S. cerevisiae only one sample after 60 minutes was taken. The results show that neither mCherry nor Mat_mCherry was taken up by A. niger. In contrast Pro_mCherry was able to infiltrate A. niger successfully.
Our results indicate that it is possible to find target-specific ligands that selectively enhance endocytosis in the aimed cell while other organisms do not even interact with them. </div>

Fluorescence microscopy

As a second proof that our ligands are specifically enhancing endocytosis in their target, we used fluorescence microscopy (Fig. 13) to show the uptake of the fusion proteins by S. cerevisiae (Fig. 14).
Fig. 13: Fluorescence microscope

Fig. 14: Fluorescence microscopy of S. cerevisiae after incubation with different fusion-proteins show specific uptake of them into the cells.
S. cerevisiae (0.35 OD) was resuspended in YPD (60 µL) and incubated (30 min, 30 °C, 450 rpm, dark) with Mat_mCherry (upper right), Opy_mCherry (lower left), Flo_mCherry (lower right) or mCherry (upper left). After washing with PBS half of the cells were visualized using a fluorescence microscope (Fig. 13) (LSM 700 (Zeiss), magnification: 100 x, filters: Texas Red [λEx=555 nm, λEm=570 nm to 800 nm], transmitted light).

It could be observed that Mat_mCherry (upper right) and Opy_mCherry (lower left) were detectable within the cells. Mat_mCherry was taken up with a slightly higher efficiency than Opy_mCherry (data not shown). In contrast Flo_mCherry (lower right) seemed to form precipitates outside the cells while the negative control mCherry without any fusion has not been taken up by S. cerevisiae.
To conclude, we can say that our selected ligands mating factor alpha and the cysteine-rich domain of Opy2 as well as a short proline-peptide were able to enhance endocytosis in the targeted cells. We also showed that Mat_mCherry is target-specific for S. cerevisiae so all in all were able to proof our concept. It is possible to enter selected target cells via cell-specific ligands.


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