Team:TU Eindhoven/Background/Membrane Anchors

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                   <h2>Membrane Anchors: OmpX and Penn Anchor</h2>
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                   <h2>Membrane Anchors: CPX and INPNC (<a href='http://parts.igem.org/Part:BBa_K811005' target="_blank">BBa K811005</a>)</h2>
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                   <p>To be able to click molecules over the entire cell surface, a protein to anchor the desired coating to the cell is needed. This protein has to be modified in such a way that it contains an azide displayed on the outside of the cell, where it can react with a DBCO-conjugate. Check also our <a href='https://2014.igem.org/Team:TU_Eindhoven/Background/SPAAC_Reaction'>SPAAC Reaction</a> Page for detailed information.</p>
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                   <p>In order to click molecules over the entire cell surface, a protein to anchor the desired coating to the cell is needed. This protein has to be modified in such a way that it contains an azide which is displayed on the outside of the cell, where it can react with a DBCO-conjugate. Check also our <a href='https://2014.igem.org/Team:TU_Eindhoven/Background/SPAAC_Reaction'>SPAAC Reaction</a> Page for detailed information.</p>
                   <h3>Membrane Anchor Protein CPX</h3>
                   <h3>Membrane Anchor Protein CPX</h3>
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<p>OmpX (Outer Membrane Protein X) is a small, monomeric β-barrel protein that is highly expressed and very useful for protein display applications in <i>E. coli</i>. CPX (Circularly Permuted OmpX) was developed as a bacterial display methodology for N- and C-terminal display. It demonstrated to enable rapid screening of very large peptide libraries with high precision and efficiency. OmpX possesses four extracellular loops, with loops 2 and 3 forming a semi rigid β-sheets protruding from the cell surface. The native N- and C-termini were fused together by a GGSG-linker, and the newly formed N- and C-termini reside on the cell surface.  This makes insertion of an unnatural amino acid fairly easy as it can be positioned before the N-terminus or after the C-terminus. By doing this, the protein itself does not have to be modified. See <a href='#Fig1'>Figure 1</a> for a schematic overview of CPX and OmpX.
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<p>CPX is a membrane protein designed for bacterial display. This membrane protein originates from OmpX. OmpX (Outer Membrane Protein x) is a small, monomeric β-barrel protein that is highly expressed and very useful for protein display applications in <i>E. coli</i>. CPX, or Circularly permutated OmpX, was developed as a bacterial display methodology for N- and C-terminal display. It is demonstrated to enable rapid screening of very large peptide libraries with high precision and efficiency. OmpX possesses four extracellular loops, with loops 2 and 3 forming semi rigid β-sheets protruding from the cell surface. The native N- and C-termini were fused together with a GGSG linker, and the newly formed N- and C-termini reside on the cell surface. See <a href='#Fig1'>Figure 1</a> for a schematic overview of CPX and OmpX. [1]
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<img id='Fig1' src="https://static.igem.org/mediawiki/2014/5/5e/TU_Eindhoven_OmpX_CPX.png" class="image_wrapper image_fr" width="1085">
<img id='Fig1' src="https://static.igem.org/mediawiki/2014/5/5e/TU_Eindhoven_OmpX_CPX.png" class="image_wrapper image_fr" width="1085">
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                   <h3>Membrane Anchor Protein INPNC</h3>
                   <h3>Membrane Anchor Protein INPNC</h3>
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<p>In 2012 the iGEM team of Penn, University of Pennsylvania, created a generalized BioBrick surface display platform, the Ice Nucleation Protein N, C termini only (INPNC), BioBrick part: <a href='http://parts.igem.org/Part:BBa_K811003'>BBa_K811003</a>.  
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<p>In 2012 the iGEM team of Penn, University of Pennsylvania, created a generalized BioBrick surface display platform, the Ice Nucleation Protein N, C termini only (INPNC), BioBrick part: <a href='http://parts.igem.org/Part:BBa_K811005' target="_blank">BBa_K811005</a>.  
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INPNC is a truncated Ice Nucleation Protein (INP, a protein which causes ice nucleation and formation but which is also used for its surface display properties. However, INP consists of an N-terminal region that is exposed to the outside membrane, as well as a central 8, 16 or 48 amino acid motif that is responsible for INP’s ice nucleation properties. These central amino acids motifs are not essential for INP’s surface display properties. Therefore INP was truncated, retaining only the N (179 aa) and C termini (49 aa) to create INPNC. The C-terminal domain is displayed at the cell surface, while the N-terminal domain remains in the outer membrane. Check also the <a href='https://2012.igem.org/Team:Penn'>Wiki Page</a> of Penn iGEM Team 2012.  
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INPNC is a truncated Ice Nucleation Protein (INP, a protein which causes ice nucleation and formation but which is also used for its surface display properties). However, INP consists of a C-terminal region that is positioned on the outer membrane, as well as a central 8, 16 or 48 amino acid motif that is responsible for INP’s ice nucleation properties. These central amino acids motifs are not essential for INP’s surface displaying properties. Therefore INP was truncated, retaining only the N (179 aa) and C termini (49 aa) to create INPNC. The C-terminal domain is displayed on the cell surface, while the N-terminal domain remains in the outer membrane. Also check the <a href='https://2012.igem.org/Team:Penn'>Wiki Page</a> of Penn iGEM Team 2012. [2]
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<h4>Bibliography</h4>
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<p>[1] Rice, J. J.. "Bacterial display using circularly permuted outer membrane protein OmpX yields high affinity peptide ligands." Protein Science 15.4 (2006): 825-836.
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[2] <a href="https://2012.igem.org/Team:Penn" target="_blank">Penn iGEM 2012</a>
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Latest revision as of 00:09, 18 October 2014

iGEM Team TU Eindhoven 2014

iGEM Team TU Eindhoven 2014

Membrane Anchors: CPX and INPNC (BBa K811005)

In order to click molecules over the entire cell surface, a protein to anchor the desired coating to the cell is needed. This protein has to be modified in such a way that it contains an azide which is displayed on the outside of the cell, where it can react with a DBCO-conjugate. Check also our SPAAC Reaction Page for detailed information.

Membrane Anchor Protein CPX

CPX is a membrane protein designed for bacterial display. This membrane protein originates from OmpX. OmpX (Outer Membrane Protein x) is a small, monomeric β-barrel protein that is highly expressed and very useful for protein display applications in E. coli. CPX, or Circularly permutated OmpX, was developed as a bacterial display methodology for N- and C-terminal display. It is demonstrated to enable rapid screening of very large peptide libraries with high precision and efficiency. OmpX possesses four extracellular loops, with loops 2 and 3 forming semi rigid β-sheets protruding from the cell surface. The native N- and C-termini were fused together with a GGSG linker, and the newly formed N- and C-termini reside on the cell surface. See Figure 1 for a schematic overview of CPX and OmpX. [1]

Figure 1. Structure of OmpX alongside a topological depiction of OmpX and CPX.

Membrane Anchor Protein INPNC

In 2012 the iGEM team of Penn, University of Pennsylvania, created a generalized BioBrick surface display platform, the Ice Nucleation Protein N, C termini only (INPNC), BioBrick part: BBa_K811005. INPNC is a truncated Ice Nucleation Protein (INP, a protein which causes ice nucleation and formation but which is also used for its surface display properties). However, INP consists of a C-terminal region that is positioned on the outer membrane, as well as a central 8, 16 or 48 amino acid motif that is responsible for INP’s ice nucleation properties. These central amino acids motifs are not essential for INP’s surface displaying properties. Therefore INP was truncated, retaining only the N (179 aa) and C termini (49 aa) to create INPNC. The C-terminal domain is displayed on the cell surface, while the N-terminal domain remains in the outer membrane. Also check the Wiki Page of Penn iGEM Team 2012. [2]

Bibliography

[1] Rice, J. J.. "Bacterial display using circularly permuted outer membrane protein OmpX yields high affinity peptide ligands." Protein Science 15.4 (2006): 825-836.

[2] Penn iGEM 2012

iGEM Team TU Eindhoven 2014