Part:BBa_K5261010
Yeast surface display pathway
We choose a yeast surface display system based on a-lectin. The a-agglutinin receptor consists of two subunits encoded by AGA1 and AGA2 genes. Aga1p is secreted extracellular and covalently binds to β-glucan on the cell wall surface. Aga2p binds to Aga1p on the N-terminus through two disulfide bonds and thus also attaches to the yeast cell surface, and its C-terminus is fused with lanthanide binding protein gene TFD. Thus, it is jointly displayed on the surface of yeast cells under the guidance of signal peptides. At the same time, short peptide sequences (Xpress™, V5, 6×His) are also present on both sides of the TFD insertion site as epitope labels, facilitating subsequent detection and characterization.
pYD1-TFD plasmid construction
The pYD1 plasmid is an expression vector designed to express, secrete and display proteins on the extracellular surface of S. cerevisiae. Gene components such as AGA2 and polyhistidine (6×His) tags have been designed on the plasmid.
Firstly, the codon-optimized TFD gene sequence of Saccharomyces cerevisiae was sent to Azenta for gene synthesis, and cloned into the position of the C terminus of the AGA2 anchor protein gene on the pYD1 plasmid vector to achieve the fusion expression of AGA2 and TFD genes (Figure 1 A and B). After receiving the strain of E. coli containing the pYD1-TFD plasmid, we amplified it, extracted the plasmid, and verified it by sequencing, which showed that the sequencing results were correct (Figure 1 C).
Figure 1. A. The pYD1-TFD plasmid. B. AGA2-TFD gene fusion expression on pYD1-TFD plasmid. C. The sequencing results showed successful insertion of the TFD gene into the pYD1 plasmid.
In addition, we used AlphaFold3 to predict the spatial structure of the Aga2-TFD fusion protein and found that the spatial structure of the TFD can still be well maintained.
Figure 2. Spatial structure of the Aga2-TFD fusion protein predicted by AlphaFold3.
pYD1-TFD, pYD1 plasmids were transformed into S. cerevisiae
The constructed pYD1-TFD plasmid and the control pYD1 plasmid were respectively transformed into Saccharomyces cerevisiae EBY100 by the lithium acetate method. The transformed yeast was coated after 2 days of culture on SC-TRP agar plates.
Figure 3. The EBY100 (pYD1) and EBY100 (pYD1-TFD) positive monoclones obtained on the SC-TRP plates.
Induce surface display protein expression
Single colonies of EBY100 (pYD1-TFD), EBY100 (pYD1), and EBY100 (as a control group) were inoculated into 5 mL of SC-TRP, SC-TRP, SC liquid medium, respectively, and cultured at 30℃ overnight. Take medium from the amplified tubes, measure absorbance at 600 nm, then inoculate to a shake flask containing 50 mL SC-TRP or SC medium, initial OD600 = 0.1, and continue for 24–36 h. Yeast was centrifuged, washed, and inoculated into the same volume of YPG liquid medium containing 2% galactose. After 48–60 h of oscillatory incubation, the displayed protein expression was predicted to reach a maximum. Cells were centrifuged and cells were resuspended in the same volume of PBS buffer and stored for 4℃.
Figure 4. Inducing yeast display protein expression in S. cerevisiae
Examined the cell surface display protein, TFD
1. SDS-PAGE test for surface display
To test whether the TFD protein was successfully displayed extracellularly, we performed SDS-PAGE electrophoresis. The Aga2-TFD fusion protein expressed in the extracellular was anchored by two disulfide bonds to the cell wall surface protein Aga1, so we could test by recovering the fusion protein Aga2-TFD from the supernatant after breaking the disulfide bond. We treated S. cerevisiae EBY100 transferred into the pYD1 plasmid and pYD1-TFD plasmid respectively with dithiothreitol (DTT) and used wild-type EBY100 as a control. After centrifugation, supernatants were removed to concentrate target proteins by ultrafiltration and verified by SDS-PAGE. While the EBY100 strain transferred into the pYD1 plasmid retained the Aga2p-Xpress Tag-V5 tag-6 × His Tag fragment (about 18.66 kDa) in the supernatant after disulfide disconnection, the EBY100 strain transferred into the pYD1-TFD plasmid retained the Aga2p-Xpress Tag-TFD-V5 tag-6 × His Tag fragment (about 54.41 kDa), while the control EBY100 strain did not have the target fragment.
Figure 5. SDS-PAGE validation of TFD proteins. The target band of EBY100 (pYD1) is 18.66 kDa, and that of EBY100 (pYD1-TFD) is 54.41 kDa.
Since we did not purify the protein, the bands on the SDS-PAGE were not obvious. However, EBY100, EBY100 transferred into pYD1 plasmid and EBY100 transferred into pYD1-TFD plasmid all showed target bands, indicating that the target protein had been successfully displayed on the surface of yeast cells.
2. Cell surface display detection by flow cell technology
Flow cytometric technology can be used to quantitatively analyze the proteins displayed on the surface of yeast cells, assessing the efficiency of protein display by combining fluorescent group-conjugated antibodies and detecting fluorescence intensity using flow cytometry. We incubated Saccharomyces cerevisiae EBY100 with the pYD1-TFD plasmid with the his tag mouse monoclonal antibody primary antibody (against the 6 × His tag on the surface-displayed protein fragment) and the fluorescent group FITC-conjugated goat anti-mouse IgG secondary antibody to label the surface-displayed protein fragments. We subsequently analyzed the fluorescence intensity on the cell surface, thus assessing the surface display level of TFD protein. Wild-type EBY100 cells were gated to distinguish populations of expressed and unexpressed surface display proteins, and the percentage of expressed cells and mean fluorescence intensity (Mean) were calculated to assess display efficiency.
Figure 6. A and B. Saccharomyces cerevisiae EBY100 cells carrying the pYD1-TFD plasmid were labeled with a 6 × His (FITC) -labeled fluorescent group-conjugated antibody and analyzed by flow cytometry.
The proportion of cells in Saccharomyces cerevisiae EBY100 with pYD1-TFD plasmid was significantly higher than the control wild-type EBY100, with the maximum display efficiency (percentage of the cells displayed) reaching 9.86% (Figure 6 A). The average fluorescence intensity (Mean) of the cell also showed the success of the surface display strain construction.(Figure 6 B)
Quantitative detection of rare earth ions adsorption by surface display strains
Having confirmed that the lanthanide-adsorbed protein TFD had been successfully demonstrated on the cell surface, we need to further test the ability of the engineered yeast to adsorb rare earth ions. Add 100 μM of TbCl3 to the EBY100 (pYD1-TFD) cell culture obtained from section 3.3. After shaking incubation for one day, centrifuge to precipitate the cells and measure the concentration of free Tb(III) in the supernatant. Cell culture medium without TbCl3 was used as a blank control. Three parallel experiments were done for each data point. Due to the high price of ICP-MS or ICP-OES, here we adopted the Arsenazo III dye-based assay method, which can be used to roughly determine the rare earth ion content in the solution, see our Protocols page for more details. The absorbance values at 650 nm can reflect the rare earth ion content within a certain range.
Figure 7. A. The absorbance at 650 nm of the solution before and after biosorption was determined by Arsenazo III assay, and obvious color depth difference was observed. B. A650 value of standard solution of TbCl3 from 0 to 150 μM.
After the adsorption process, the TbCl3 content in the solution decreased significantly, which was manifested in the obvious color depth difference of the Arsenazo III – REE complex formed before and after adsorption (Figure 7 A), which was also reflected by the value of A650. However, due to the limitation of the Arsenazo III assay and the extremely low concentration of Tb(Ⅲ) ions in the solution after adsorption, we could not quantitatively detect the exact concentration of rare earth ions by drawing a standard curve. However, we could roughly estimate it by the "two-point interval method" according to the value of A650: For the solution after adsorption, three parallel data points were measured in a microplate reader, and the A650 obtained was between that of 2 μM and 10 μM TbCl3 standard solution (Figure 7 B), so it could be preliminarily judged that c(Tb3+) in the solution after the adsorption was also in this range. Since we initially added c(Tb3+) = 100 μM (before adsorption), it could be considered that our surface display strain had a strong adsorption capacity for rare earth ions.
Outlook
Due to time constraints, we only used S. cerevisiae strain EBY100 for proof of concept in the experiments of this module, with preliminary demonstration that TFD protein display on the yeast cell surface can construct a whole-cell bio-adsorbent with strong biosorption capacity for rare earth ions. However, in future industrial applications, we expect to integrate AGA2-linker-TFD gene expression cassette into the Issatchenkia orientalis genome to achieve constitutive expression to achieve more stable and continuous expression of lanthanide binding protein TFD, and thus better application to bio-adsorption in acid rare earth mine wastewater.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 273
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 736
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 1404
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