Difference between revisions of "Part:BBa K5166001"
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<h1>Usage</h1>
 
<h1>Usage</h1>
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<p>&nbsp;&nbsp;&nbsp;&nbsp;The BIT-China team constructed a yeast surface display 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.</p><br><br>
 +
<p>&nbsp;&nbsp;&nbsp;&nbsp;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.</p>
  
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<p>&nbsp;&nbsp;&nbsp;&nbsp;Nickel-binding peptide 2 (NBP2) is a nickel-binding peptide. It can bind Ni(II) specificaly. We display it with <i>Pichia pastoris</i> through cell-surface display systems.
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<h1>Construction</h1>
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</p>
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<p>&nbsp;&nbsp;&nbsp;&nbsp;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 respectively inserting 10 metal binding peptide genes as target genes between linker and Pir1p, we obtained corresponding surface display plasmids.</p>
 +
<p>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.</p><br>
  
 +
<h1>Proof of Surface Display and Display Effect</h1>
 +
<p>&nbsp;&nbsp;&nbsp;&nbsp;Because the system contains Myc-tag, it can be detected by indirect immunofluorescence method. If it is successfully displayed on the surface of the cell, it can be observed that there is obvious red fluorescence on the surface; conversely, if there is no obvious red fluorescence on the surface, it is not successfully displayed. The samples were prepared by indirect yeast immunofluorescence method and examined by fluorescence microscopy. The following figures (a) and (b) showed that GS115 in the control group had no fluorescence, while CBP1-pir cells had strong fluorescence and a ring around the cell edge. Pichia pastoris GS115 was a sphere, and the surface display peptides was evenly distributed on the cell surface, and the cell edge was concentrated, so the cell edge brightness was the highest, indicating a successful display.</p><br>
 +
<p>&nbsp;&nbsp;&nbsp;&nbsp;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. 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.</p><br>
 +
<p>&nbsp;&nbsp;&nbsp;&nbsp;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. (c) and (d) show strains showing BH2807 on the surface. FIG. (e) and (f) show the strains showing mntR on the surface; Figures (g) and (h) show the strains of TssS on the surface. The roughness of cell surface was found in the order of mntR-pir > TssS-pir > BH2807-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.</p><br>
  
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<h1>Biology</h1>
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<h1>Reference</h1>
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<p>&nbsp;&nbsp;&nbsp;&nbsp;NBP2 is selected from Phage Display Library Screening: The Ph.D.-12 peptide library kit (E8110S, New England Bio Labs, USA). A study has shown that the sorption of nickel ions on the surface of yeast cells increased with the increasing number of nickel Ni(II)-binding peptides displayed. The combined expression of the three peptides(NBP1+NBP2+NBP3) by <i>EBY100/pYD1-N123</i> demonstrated the highest sorption of Ni(II) (2.603 ± 0.004 g g−1, dry weight) and an enhanced sorption capacity of 60.15%, compared to <i>S. cerevisiae EBY100</i>[1].</p>
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<p>[1] SK Amir Hossain;SM Rifat Rahman;Toufiq Ahmed;Chanchal Mandal.An overview of yeast cell wall proteins and their contribution in yeast display system[J].Asian Journal of Medical and Biological Research,2019,Vol.5(4): 246-257<br>（https://www.researchgate.net/publication/339288607_An_overview_of_yeast_cell_wall_proteins_and_their_contribution_in_yeast_display_system）<br>
 +
[2] Andreu, C.1;del Olmo, M.2.Yeast arming systems: pros and cons of different protein anchors and other elements required for display(Review)[J].Applied Microbiology and Biotechnology,2018,Vol.102(6): 2543-2561<br>（https://link.springer.com/article/10.1007/s00253-018-8827-6）<br>
 +
[3] Tabanag, IDF (Tabanag, Ian Dominic Flormata)1;Chu, IM (Chu, I-Ming)2;Wei, YH (Wei, Yu-Hong)3;Tsai, SL (Tsai, Shen-Long)1.The Role of Yeast-Surface-Display Techniques in Creating Biocatalysts for Consolidated BioProcessing[J].Catalysts,2018,Vol.8(3): 94<br>（https://www.mdpi.com/2073-4344/8/3/94）<br>
 +
[4] Kouichi Kuroda;Ken Matsui;Shinsuke Higuchi;Atsushi Kotaka;Hiroshi Sahara;Yoji Hata;Mitsuyoshi Ueda.Enhancement of display efficiency in yeast display system by vector engineering and gene disruption.[J].Applied Microbiology & Biotechnology,2009,Vol.82(4): 713-719<br>
 +
（https://pubmed.ncbi.nlm.nih.gov/19123001/）<br>
 +
[5] Tanaka, Tsutomu 1;Matsumoto, Sayoko 1;Yamada, Mari 1;Yamada, Ryosuke 2;Matsuda, Fumio 2;Kondo, Akihiko 1.Display of active beta-glucosidase on the surface of Schizosaccharomyces pombe cells using novel anchor proteins.[J].Applied Microbiology & Biotechnology,2013,Vol.97(10): 4343-4352<br>（https://link.springer.com/article/10.1007/s00253-013-4733-0）</p><br>
  
  
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<h1>Simulation</h1>
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<!-- Add more about the biology of this part here
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<p> &nbsp;&nbsp;&nbsp;&nbsp;We used the MLatom calculation program on the XACS platform to perform structural calculations of the binding between metal ion binding peptides and metal ions[2-6], in order to predict the binding ability.
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===Usage and Biology===
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In the following video: white is H, gray is C, blue is N, red is O, flesh color is Co, purple is Li, light green is Cl, dark green is Ni.<br>
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(1) Sequence<br>
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&nbsp;&nbsp;&nbsp;&nbsp;Here we perform machine learning quantum chemistry calculations for the sequence NBP2: HAVSPTLPAYSK. Firstly, the geometric structure of the polypeptide chain was optimized, and the folded configuration was obtained, as shown below.<br>
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(2) Structural analysis of single ion binding<br>
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&nbsp;&nbsp;&nbsp;&nbsp;We simulated the binding of a single Ni ion to a folded polypeptide chain. Through structural optimization, we get the following results:<br><br>
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&nbsp;&nbsp;&nbsp;&nbsp;It can be seen that the atoms coordinating with Ni2+ are mainly O and N atoms on the polypeptide chain. The Ni2+ ion is encased.<br>
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(3) The combination of multiple ions<br>
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&nbsp;&nbsp;&nbsp;&nbsp;We designed this metal-binding peptide in the hope that they could trap multiple metal ions and increase efficiency. In order to analyze the binding of multiple Ni2+ ions, four Ni2+ ions were added to the molecular model, and four Cl- ions were added to balance their charge, with a total charge of +4. Through structural optimization, we get the following results:<br>
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&nbsp;&nbsp;&nbsp;&nbsp;It can be seen that the N and carbonyl O atoms of higher amines participate in the coordination, the counterion Cl- participates in the coordination, and the counterion also interacts with the carbon skeleton, which can regulate the overall charge distribution of the polypeptide chain, regulate the structure of the polypeptide, and stabilize the system.</p>
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<h1>Experiments</h1>
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<p>1.We used <i>Pichia Pastoris</i> GS115 as chassis cell and pGAPZα plasmid to design the display system. By inserting the NBP2 metal-binding peptide gene as the target gene, we obtained the corresponding surface display plasmid.<br>
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2.After constructing the plasmid, we introduced it into <i>Escherichia coli</i> for amplification. After amplification, the plasmids were extracted and purified, and sent for sequencing. After obtaining the correct sequencing results, the plasmid was transformed into <i>Pichia pastoris</i> by means of electrical stimulation. Finally, by colony PCR, we determined that the plasmid was successfully introduced into the yeast.<br>
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3.After obtaining the engineered yeast, we designed some experimental schemes to qualitatively test their adsorption effect on target metal ions. In order to test the adsorption effect, the engineered yeast and the prepared single metal solution were mixed according to a certain proportion, removed and centrifuged after 2 hours, and the supernatant and precipitation were stored respectively. We chose to use a graphite furnace to detect the concentration of metal ions in the supernatant. 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 strain on the target metal ions, and made a comparison to select the strain with better adsorption effect.</p>
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<h1>Reference</h1>
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<p>[1]Li H, Dong W, Liu Y, et al. Enhanced Biosorption of Nickel Ions on Immobilized Surface-Engineered Yeast Using Nickel-Binding Peptides[J]. Front Microbiol, 2019,10:1254.<br>
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[2]Jmol: an open-source Java viewer for chemical structures in 3D. http://www.jmol.org/<br>
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[3] Q. Sun, et al. J. Chem. Phys. 2020, 153, 024109<br>
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[4] Q. Sun, et al. WIREs Comput. Mol. Sci. 2018, 8, e1340<br>
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[5] Q. Sun, J. Comp. Chem. 2015, 36, 1664<br>
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[6] L.-P. Wang, C. C. Song, J. Chem. Phys. 2016, 144, 214108</p>
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<h1>Sequence and Features</h1>
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<partinfo>BBa_K5166001 SequenceAndFeatures</partinfo>
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K5166032 SequenceAndFeatures</partinfo>
  
  
 
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===Functional Parameters===
 
===Functional Parameters===
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<partinfo>BBa_K5166001 parameters</partinfo>
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<partinfo>BBa_K5166032 parameters</partinfo>
 
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Revision as of 13:38, 30 September 2024

Pichia Pastoris GS115 Surface Display System

Usage

    The BIT-China team constructed a yeast surface display 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 respectively inserting 10 metal binding peptide genes as target genes between linker and Pir1p, we obtained corresponding surface display plasmids.

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.


Proof of Surface Display and Display Effect

    Because the system contains Myc-tag, it can be detected by indirect immunofluorescence method. If it is successfully displayed on the surface of the cell, it can be observed that there is obvious red fluorescence on the surface; conversely, if there is no obvious red fluorescence on the surface, it is not successfully displayed. The samples were prepared by indirect yeast immunofluorescence method and examined by fluorescence microscopy. The following figures (a) and (b) showed that GS115 in the control group had no fluorescence, while CBP1-pir cells had strong fluorescence and a ring around the cell edge. Pichia pastoris GS115 was a sphere, and the surface display peptides was evenly distributed on the cell surface, and the cell edge was concentrated, so the cell edge brightness was the highest, indicating a successful display.


    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. 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.


    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. (c) and (d) show strains showing BH2807 on the surface. FIG. (e) and (f) show the strains showing mntR on the surface; Figures (g) and (h) show the strains of TssS on the surface. The roughness of cell surface was found in the order of mntR-pir > TssS-pir > BH2807-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.


Reference

[1] SK Amir Hossain;SM Rifat Rahman;Toufiq Ahmed;Chanchal Mandal.An overview of yeast cell wall proteins and their contribution in yeast display system[J].Asian Journal of Medical and Biological Research,2019,Vol.5(4): 246-257
（https://www.researchgate.net/publication/339288607_An_overview_of_yeast_cell_wall_proteins_and_their_contribution_in_yeast_display_system）
[2] Andreu, C.1;del Olmo, M.2.Yeast arming systems: pros and cons of different protein anchors and other elements required for display(Review)[J].Applied Microbiology and Biotechnology,2018,Vol.102(6): 2543-2561
（https://link.springer.com/article/10.1007/s00253-018-8827-6）
[3] Tabanag, IDF (Tabanag, Ian Dominic Flormata)1;Chu, IM (Chu, I-Ming)2;Wei, YH (Wei, Yu-Hong)3;Tsai, SL (Tsai, Shen-Long)1.The Role of Yeast-Surface-Display Techniques in Creating Biocatalysts for Consolidated BioProcessing[J].Catalysts,2018,Vol.8(3): 94
（https://www.mdpi.com/2073-4344/8/3/94）
[4] Kouichi Kuroda;Ken Matsui;Shinsuke Higuchi;Atsushi Kotaka;Hiroshi Sahara;Yoji Hata;Mitsuyoshi Ueda.Enhancement of display efficiency in yeast display system by vector engineering and gene disruption.[J].Applied Microbiology & Biotechnology,2009,Vol.82(4): 713-719
（https://pubmed.ncbi.nlm.nih.gov/19123001/）
[5] Tanaka, Tsutomu 1;Matsumoto, Sayoko 1;Yamada, Mari 1;Yamada, Ryosuke 2;Matsuda, Fumio 2;Kondo, Akihiko 1.Display of active beta-glucosidase on the surface of Schizosaccharomyces pombe cells using novel anchor proteins.[J].Applied Microbiology & Biotechnology,2013,Vol.97(10): 4343-4352
（https://link.springer.com/article/10.1007/s00253-013-4733-0）



Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 751
    Illegal NotI site found at 1240
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 730
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 1360
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 238