Part:BBa_K5166040

pGAPZα-mntR-pir

Usage and Biology

The BIT-China team constructed a yeast surface display system that could link target proteins to anchor proteins to be displayed on the surface of Pichia pastoris. Uses of the system include:
(1)For high efficiency protein preparation: displaying specific proteins on the surface of strain can not only improve the stability of the proteins, but also facilitate the directed evolution and optimization of the proteins.
(2)Construction of whole cell adsorbents: capture by binding of surface display proteins to specific substances, such as metal binding peptides on the surface to adsorb metal ions.
(3)Manufacturing biosensors: immobilization of biorecognition elements to create biosensors with high sensitivity and specificity.
Yeast surface display technology is a method to fuse the target protein with the anchor protein and make it stably attached to the yeast cell surface, so as to display the foreign protein on the yeast cell wall. In this system, Pichia pastoris GS115 was used as chassis cell, and the target protein was displayed by Pir anchoring protein of Pir anchoring system. In our experiments, we selected metal-binding peptides as target proteins for display in order to obtain engineered yeasts capable of selective adsorption of metal ions.

Construction

Through investigation, we found that the anchor motif Pir1 performed well in the surface display protein in Pichia pastoris. Therefore, we link Pir1 to the target protein via a linker (3×SerGly) and initiate peptides using signal peptide α-factor. In addition, in order to facilitate the subsequent detection of surface display protein content, we added a Myc-tag to the N-terminal of Pir1, allowing an immunofluorescence assay to characterize the displayed peptides. We choose GAP promoter as the promoter and AOX terminator as the terminator to make these sequences efficiently expressed. By inserting mntR genes as target genes between linker and Pir1p, we obtained corresponding surface display plasmids.
mntR is a DtxR family metalloregulators from Mycobacterium tuberculosis. It can bind Mn(Ⅱ) specificaly.
After the plasmid construction was completed, we introduced it into Escherichia coli DH5α for amplification. After amplification, the plasmids were extracted and purified, and sent for sequencing. After obtaining the correct sequencing results, the recombinant plasmids were linearized using the Bln I restriction site, following which they were integrated into the PGAP locus of Pichia Ppastoris GS115 genome, respectively. Finally, by colony PCR, we determined that the plasmid was successfully introduced into the yeast.

Fig. 1 Electrophoretic map of plasmids containing Metal-binding peptides gene.

Fig. 2 Electrophoretic map of engineered yeast after colony PCR.

Proof of Surface Display and Display Effect

Considering that the target protein shown is a metal binding peptide, the adsorption effect of the engineered strain on the target metal ions can be qualitatively tested to verify the effectiveness of the surface display system. We mixed the fermentation broth of the engineered strain and the prepared single metal solution in a certain proportion, took them out and centrifuged them 2 hours later, and stored the supernatant and precipitated them respectively.
1.Due to the high concentration of metal ions in the supernuant after adsorption, it is necessary to dilute it with deionized water and dilute nitric acid, and then test the sample with appropriate concentration by graphite furnace. By calculating the ratio of the reduced metal concentration in the supernatant to the original added metal concentration, we obtained the adsorption rate of the engineered strains to the target metal ion, so as to reflect the effect of surface display.
2.Yeast cells after adsorption was completed were used for electron microscopy sampling, and we used a scanning electron microscope to observe yeast cells exhibiting metal-binding peptides on their surfaces.Figure (a) and (b) are the control group GS115; FIG. (e) and (f) show the strains showing mntR on the surface. The roughness of cell surface was found in the order of mntR-pir > GS115. Through literature research, we believe that the degree of cell surface roughnes is related to the number of displayed proteins, the more display proteins the rougher the cell surface. On the one hand, this directly shows the effectiveness of the surface display system, and on the other hand, it confirms the comparison of adsorption effects of engineered strains to a certain extent.

Fig. 3The adsorption rate of mntR-pir, TssS-pir and BH2807-pir for Mn²⁺ within 2 hours.

Fig. 4Scanning electron microscope images of yeast strains. a) GS115 (×25000 times); b) GS115 (×40000 times);e) mntR-pir (×25000 times); f) mntR-pir(×40000 times).

Reference

[1]Hossain, S. A., Rahman, S. R., Ahmed, T., & Mandal, C. (2020). An overview of yeast cell wall proteins and their contribution in yeast display system. Asian Journal of Medical and Biological Research, 5(4), 246-257. doi: 10.3329/ajmbr.v5i4.45261
[2]Andreu, C., & del Olmo, M. (2018). Yeast arming systems: pros and cons of different protein anchors and other elements required for display. APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 102(6), 2543-2561. doi: 10.1007/s00253-018-8827-6
[3]Tabañag, I., Chu, I., Wei, Y., & Tsai, S. (2018). The Role of Yeast-Surface-Display Techniques in Creating Biocatalysts for Consolidated BioProcessing. CATALYSTS, 8(3). doi: 10.3390/catal8030094
[4] Kuroda, K., Matsui, K., Higuchi, S., Kotaka, A., Sahara, H., Hata, Y.,... Ueda, M. (2009). Enhancement of display efficiency in yeast display system by vector engineering and gene disruption. APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 82(4), 713-719. doi: 10.1007/s00253-008-1808-4
[5] Tanaka, T., Matsumoto, S., Yamada, M., Yamada, R., Matsuda, F.,... Kondo, A. (2013). Display of active beta-glucosidase on the surface of Schizosaccharomyces pombe cells using novel anchor proteins. APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 97(10), 4343-4352. doi: 10.1007/s00253-013-4733-0

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal XbaI site found at 1677
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 1174
  Illegal NotI site found at 1663
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 730
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal XbaI site found at 1677
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal XbaI site found at 1677
  Illegal AgeI site found at 1783
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 238