
Part:BBa_K2926049
Mating factor alpha from S. cerevisiae fused to mCherry
Mating factor alpha from S. cerevisiae was N-terminally fused to the red fluorescent protein mCherry to enable visualization of the protein.
Contents
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. Mating factor alpha specifically binds the mating pheromone receptor Ste2 that is taken up into the cell upon binding to the pheromone (Bardwell 2004).
Flo_mCherry was characterized together with the three other fusion-proteins Flo_mCherry (BBa_K2926050), Opy_mCherry (BBa_K2926051) and Pro_mCherry (BBa_K2926068).
Sequence and Features
Sequence was validated by Sanger sequencing.
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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).

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.

Expression cultures were harvested via centrifugation for 20 min at 4 °C and 4 000 rpm.

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.

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.

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.

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.
Protein characterization

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.

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.

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.
Endocytosis assays
Fluorescence in the supernatant

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.

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 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).

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.
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


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).
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.
References
Bardwell, Lee (2004): A walk-through of the yeast mating pheromone response pathway. In: Peptides 25 (9).
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